MODIFIED Cry1Ca TOXINS USEFUL FOR CONTROL OF INSECT PESTS

ABSTRACT

The subject invention concerns Bacillus thuringiensis modified Cry1Ca insecticidal toxins and the polynucleotide sequences which encode these toxins. Uses in transgenic plants are described as are methods for protecting crops from insect pest damage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/780,501, filed Feb. 3, 2020, which iscontinuation application of U.S. patent application Ser. No. 15/541,303,filed Jun. 30, 2017, now U.S. Pat. No. 10,570,182, which is the U.S.National Phase application of International Patent Application numberPCT/US15/066182, filed Dec. 16, 2015, which claims the benefit of U.S.Provisional patent application No. 62/097,833, filed Dec. 30, 2014, thedisclosures of each of which are incorporated herein by reference intheir entireties.

REFERENCE TO A SEQUENCE LISTING

The official copy of the sequence listing is submitted electronicallyvia USPTO Patent Center as an XML formatted sequence listing with a filenamed 70951-US-PCN2 created on Sep. 18, 2022 and having a size of104,220 bytes and is filed concurrently with the specification. Thesequence listing comprised in this XML formatted document is part of thespecification and is herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

The subject invention concerns modification of a Bacillus thuringiensispesticidal toxin, the polynucleotide sequences which encode these toxinsand transgenic plants that produce these toxins.

BACKGROUND OF DISCLOSURE

Insects and other pests cost farmers billions of dollars annually incrop losses and expense to keep these pests under control. In additionto losses in field crops, insect pests are also a burden to vegetableand fruit growers, to producers of ornamental flowers, and to homegardeners. The losses caused by insect pests in agricultural productionenvironments include decrease in crop yield, reduced crop quality, andincreased harvesting costs.

Insect pests are mainly controlled by intensive applications of chemicalpesticides, which are active through inhibition of insect growth,prevention of insect feeding or reproduction, or cause death. Goodinsect control can thus be reached, but these chemicals can sometimesaffect other beneficial insects. Another problem resulting from the wideuse of chemical pesticides is the appearance of resistant insectpopulations. This has been partially alleviated by various resistancemanagement practices, but there is an increasing need for alternativepest control agents. Biological pest control agents, such as Bacillusthuringiensis (B.t.) strains expressing pesticidal toxins likedelta-endotoxins, have also been applied to crop plants withsatisfactory results, offering an alternative or compliment to chemicalpesticides. The genes coding for some of these delta-endotoxins havebeen isolated and their expression in heterologous hosts have been shownto provide another tool for the control of economically important insectpests. In particular, the expression of insecticidal toxins, such asBacillus thuringiensis delta-endotoxins, in transgenic plants haveprovided efficient protection against selected insect pests, andtransgenic plants expressing such toxins have been commercialized,allowing farmers to reduce applications of chemical insect controlagents.

Lepidopterans are an important group of agricultural, horticultural, andhousehold pests which cause a large amount of damage each year. Thisinsect order encompasses foliar- and root-feeding larvae and adults.Lepidopteran insect pests include, but are not limited to: Achoroiagrisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotisipsilon (black cutworm “BCW”), Alabama argillacea, Alsophila pometaria,Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisotasenatoria, Antheraea pernyi, Anticarsia gemmatalis (velvetbeancaterpillar “VBC”), Archips sp., Argyrotaenia sp., Athetis mindara,Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneurasp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydialatiferreanus, Cydia pomonella, Datana integerrima, Dendrolimussibericus, Desmia feneralis, Diaphania hyalinata, Diaphania nitidalis,Diatraea grandiosella (southwestern corn borer “SWCB”), Diatraeasaccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella,Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoelliaambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoamessoria, Galleria mellonella, Grapholita molesta, Harrisina americana,Helicoverpa subflexa, Helicoverpa zea (corn earworm “CEW”), Heliothisvirescens (tobacco budworm “TBW”), Hemileuca oliviae, Homoeosomaelectellum, Hyphantia cunea, Keiferia lycopersicella, Lambdinafiscellaria fiscellaria, Lambdinafiscellaria lugubrosa, Leucoma salicis,Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macallathyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurata,Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchrapicta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis (Europeancorn borer “ECB”), Paleacrita vernata, Papiapema nebris (common stalkborer), Papilio cresphontes, Pectinophora gossypiella, Phryganidiacalfornica, Phyllonorycter blancardella, Pieris napi, Pieris rapae,Plathypena scabra, Platynotaflouendana, Platynota stultana, Platyptiliacarduidactyla, Plodia interpunctella, Plutella xylostella (diamondbackmoth “DBM”), Pontia protodice, Pseudaletia unipuncta, Pseudoplusiaincludens (soybean looper “SBL”), Sabulodes aegrotata, Schizuraconcinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera eridania(southern armyworm “SAW”), Spodoptera frugiperda (fall armyworm “FAW”),Spodoptera exigua (beet armyworm “BAW”), Thaurnstopoea pityocampa,Ensola bisselliella, Trichoplusia ni (cabbage looper “CL”), Udearubigalis, Xylomyges curiails, and Yponomeuta padella. Any genus listedabove (and others), generally, can also be targeted as a part of thesubject invention. Any additional insects in any of these genera (astargets) are also included within the scope of this invention.

Bacillus thuringiensis (B.t.) is a soil-borne, Gram-positive, sporeforming bacterium that produces insecticidal crystal proteins known asdelta endotoxins or Cry proteins (reviewed in Schnepf et al., 1998).Novel Crystal (Cry) proteins with new insecticidal properties continueto be discovered at an increasing rate, and over 440 Cry genes have beenreported. Currently, there are over 450 unique Cry and Cytotoxin (Cyt)proteins classified among 57 primary homology ranks. Cry proteins arenamed based on the degree of sequence identity, with primary, secondaryand tertiary boundaries occurring at approximately 45%, 78% and 95%identity, respectively; close alleles are assigned new quaternarydesignations (Crickmore et al., 1998). An expansive list of deltaendotoxins is maintained and regularly updated athttp://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html. Thereare currently over 73 main groups of “Cry” toxins (Cry1-Cry73), withadditional Cyt toxins and Vegetative Insecticidal Protein (VIP) toxinsand the like. Many of each numeric group have capital-letter subgroups,and the capital letter subgroups have lower-cased letter sub-subgroups.(Cry1 has A-L, and Cry1A has a-i, for example).

B.t. proteins have been used to create the insect-resistant transgenicplants that have been successfully registered or deregulated andcommercialized to date. These include Cry1Ab, Cry1Ac, Cry1F, Vip3A,Cry34Ab1/Cry35Ab1, and Cry3Bb in corn, Cry1Ac, Vip3A and Cry2Ab incotton, and Cry3A in potato. B.t. toxins represent over 90% of thebioinsecticide market and essentially the entire source of genes fortransgenic crops that have been developed to provide resistance toinsect feeding.

Cry proteins are oral intoxicants that function by acting on midgutcells of susceptible insects. The active forms of many Cry proteinscomprise three distinct protein domains. The most well studied B.t.proteins are members of the three-domain Cry delta-endotoxins. Theseproteins range in size from approximately 70 kDa to 130 kDa. Primaryprotein sequence analysis reveals five highly conserved sequence blocksand a high degree of sequence variability between conserved blocks threeand five (Schnepf et al., 1998).

Three dimensional crystal structures have been determined for Cry1Aa1,Cry2Aa1, Cry3Aa1, Cry3Bb1, Cry4Aa, Cry4Ba and Cry8Ea1 as examples. Thesestructures are remarkably similar and are comprised of three distinctdomains with the following features (reviewed in de Maagd et al., 2003).Domain I is a bundle of seven alpha helices where helix five issurrounded by six amphipathic helices. This domain has been implicatedin midgut membrane insertion and pore formation. It shares homology withother pore forming proteins including hemolysins and colicins. Domain IIis comprised of three anti-parallel beta sheets packed together in abeta prism. This domain shares homology with certaincarbohydrate-binding proteins including vitelline and jacaline. Theloops of this domain play important roles in binding insect midgutreceptors. In Cry1A proteins, surface exposed loops at the apices ofdomain II beta sheets are involved in binding to lepidopteran cadherinreceptors. Domain III is a beta sandwich structure that interacts with asecond class of receptors, examples of which are aminopeptidase andalkaline phosphatase in the case of Cry1A proteins (Piggot and Ellar,2007). Structurally this domain is related to carbohydrate-bindingdomains of proteins such as glucanases, galactose oxidase, sialidase andothers. This domain binds certain classes of receptor proteins andperhaps participates in insertion of an oligomeric toxin pre-pore.Conserved B.t. sequence blocks 2 and 3 map near the N-terminus andC-terminus of domain 2, respectively. Hence, these conserved sequenceblocks 2 and 3 are approximate boundary regions between the threefunctional domains. These regions of conserved DNA and protein homologyhave been exploited for engineering recombinant B.t. toxins (U.S. Pat.No. 6,090,931, WO 91/01087, WO95/06730, WO 1998022595).

One proposed model for Cry protein mode of action is based on poreformation in the midgut membranes of susceptible insects (Knowles andEllar, 1987). In the current version of this model (Bravo et al., 2007),binding to both cadherin and aminopeptidase receptors on Lepidopteranmidgut membranes are required for Cry protein toxicity. According to thepore formation model, Cry protein intoxication involves severalsteps: 1) Proteolytic processing of soluble Cry protoxin to an activatedcore toxin; 2) Cry protein binding to cadherin receptors on the insectmidgut; 3) further proteolytic cleavage at the core toxin N-terminus toremove an α-helical region; 4) Cry protein oligomerization to form apre-pore; 5) pre-pore binding to second site membrane receptors(aminopeptidases and alkaline phosphatases); 6) pre-pore insertion intothe membrane and 7) osmotic cell lysis leading to midgut disruption andinsect death.

The widespread adoption of insect-resistant transgenic plant technologygives rise to a concern that pest populations will develop resistance tothe insecticidal proteins produced by these plants. Several strategieshave been suggested for preserving the utility of B.t.-based insectresistance traits which include deploying proteins at a high dose incombination with a refuge, and alternation with, or co-deployment of,different toxins (McGaughey et al. (1998), “B.t. Resistance Management,”Nature Biotechnol. 16:144-146).

The development of insect resistance to B.t. Cry proteins can resultthrough several mechanisms (Heckel et al., 2007, Piggot and Ellar,2007). Multiple receptor protein classes for Cry proteins have beenidentified within insects, and multiple examples exist within eachreceptor class. Resistance to a particular Cry protein may develop, forexample, by means of a mutation within the toxin-binding portion of acadherin domain of a receptor protein. A further means of resistance maybe mediated through a protoxin-processing protease. Thus, resistance toCry1A toxins in species of Lepidoptera has a complex genetic basis, withat least four distinct, major resistance genes. Lepidopteran insectsresistant to Cry proteins have developed in the field for Plutellaxylostella (Tabashnik, 1994), Trichoplusia ni (Janmaat and Myers 2003,2005), Helicoverpa zea (Tabashnik et al., 2008), and Spodopterafrugiperda (Storer, et al., 2010). Development of new high potency Cryproteins will provide additional tools for management of Lepidopteraninsect pests.

This invention provides B.t. insecticidal proteins that are effective incontrolling insects that are resistant to Cry1Ac and Cry1F. Theseprotein toxins may be used advantageously to protect agronomic cropsfrom insect feeding damage. The ability to express these insect toxinsin such a manner that sufficient quantity of the functionally activeprotein is present in a crop of interest is also a subject of thisinvention.

BRIEF SUMMARY OF THE INVENTION

A modified Cry1Ca toxin comprising residues 2 to 68 of SEQ ID NO:2wherein amino acid residue 54 is chosen from the group consisting of Glyand Ala, amino acid residue 57 is chosen from the group consisting ofLeu and Met, and amino acid residue 68 is chosen from the groupconsisting of Val, Phe, and Ile. A modified Cry1Ca toxin comprisingresidues 2 to 628 of SEQ ID NO:210 wherein amino acid residue 54 ischosen from the group consisting of Gly and Ala, amino acid residue 57is chosen from the group consisting of Leu and Met, amino acid residue68 is chosen from the group consisting of Val, Phe, and Ile, amino acidresidue 73 is chosen from the group consisting of Trp, Ala and Met,amino acid residue 596 is chosen from the group consisting of Phe, Metand Ala, and amino acid residue 620 is chosen from the group consistingof Leu and Phe. The modified Cry1Ca toxins of the foregoing furthercomprising a carboxy terminal extension consisting of amino acidresidues 629 to 1164 of SEQ ID NO:36. The modified Cry1Ca toxins of theforegoing further comprising a carboxy terminal extension consisting ofamino acid residues 629 to 1164 of SEQ ID NO:36. The modified Cry1Catoxins of the foregoing further comprising an amino terminal extensionconsisting of amino acid residues 1 to 74 of SEQ ID NO:40. The modifiedCry1Ca toxins of the foregoing further comprising an amino terminalextension consisting of amino acid residues 1 to 74 of SEQ ID NO:40. Themodified Cry1Ca toxins of the foregoing further comprising an aminoterminal extension consisting of amino acid residues 1 to 74 of SEQ IDNO:40. The modified Cry1Ca toxins of the foregoing further comprising anamino terminal extension consisting of amino acid residues 1 to 74 ofSEQ ID NO:40.

DNA encoding modified Cry1Ca toxins, transgenic plants producingmodified Cry1Ca toxins and methods of controlling insect pests using themodified Cry1Ca toxins are included in the invention.

The subject invention concerns novel materials and methods forcontrolling arthropod pests that are detrimental to plants and toagriculture. In a preferred embodiment, the subject invention providesmaterials and methods for the control of Lepidopteran pests.

Specific B.t. Cry proteins (endotoxins, toxins) useful according to theinvention include toxins which can be obtained from the B.t. isolatedesignated as MR-1206. The subject invention also includes the use ofmutants of the exemplified B.t. isolate and toxins which have improvedLepidopteran-active properties, that resist protease processing, orexpress at high levels when the genes are transformed into aheterologous expression system. Procedures for making mutants are wellknown in the microbiological art. Ultraviolet light and chemicalmutagens such as nitrosoguanidine are used extensively toward this end.

The subject protein toxins can be “applied” or provided to contact thetarget insects in a variety of ways. For example, transgenic plants(wherein the protein is produced by and present in the plant) can beused and are well-known in the art. Expression of the toxin genes canalso be achieved selectively in specific tissues of the plants, such asthe roots, leaves, etc. This can be accomplished via the use oftissue-specific promoters, for example. Spray-on applications areanother example and are also known in the art. The subject proteins canbe appropriately formulated for the desired end use, and then sprayed(or otherwise applied) onto the plant and/or around the plant and/or tothe vicinity of the plant to be protected, before an infestation isdiscovered, after target insects are discovered, both before and after,and the like. The subject protein can also be appropriately formulatedand applied to the seeds as a seed treatment that allows the protein tobe in contact with the root area of the plant to protect it from rootfeeding insects. Bait granules, for example, can also be used and areknown in the art.

The subject proteins can be used to protect practically any type ofplant from damage by a Lepidopteran insect. Examples of such plantsinclude maize, sunflower, soybean, cotton, canola, rice, sorghum, wheat,barley, vegetables, ornamentals, peppers (including hot peppers), sugarbeets, fruit, and turf grass, to name but a few. Especially preferredplants are maize, soybean and cotton. A most preferred plant is maize.Another most preferred plant is soybean. Another most preferred plant iscotton.

In one embodiment of the subject invention, the polynucleotide sequencesof the subject invention encode toxins of approximately 68-71 kDa. Thesetoxins are used to control Lepidopteran pests, especially fallarmyworms, diamondback moths, southwestern corn borer, southernarmyworm, corn earworm, and European corn borer. In a preferredembodiment, the subject invention concerns plants cells transformed withat least one polynucleotide sequence of the subject invention such thatthe transformed plant cells produce and contain pesticidal toxins of theinvention in tissues consumed by the target pests.

Alternatively, the B.t. isolate of the subject invention, or recombinantmicrobes expressing genes encoding the pesticidal toxin proteinsdescribed herein, can be used to control insect pests. In this regard,the invention includes the treatment of substantially intact B.t. cells,and/or recombinant cells containing the toxins of the invention, treatedto prolong the pesticidal activity when the substantially intact cellsare applied to the environment of a target pest. The treated cell actsas a protective coating for the pesticidal toxin. The toxin becomesactive upon ingestion by a target insect.

One aspect of the invention pertains to isolated nucleic acid moleculescomprising nucleotide sequences encoding pesticidal proteins andpolypeptides or biologically active portions thereof, as well as nucleicacid molecules sufficient for use as hybridization probes to identifynucleic acids encoding the claimed toxins. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g. cDNAor genomic DNA) and RNA molecules (e.g. mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

Nucleotide sequences encoding the proteins of the present inventioninclude the sequences set forth in SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and complements thereof.By “complement” is intended a nucleotide sequence that is sufficientlycomplementary to a given nucleotide sequence such that it can hybridizeto the given nucleotide sequence to thereby form a stable duplex(double-stranded) molecule. The corresponding amino acid sequences forthe pesticidally active modified Cry1Ca toxins encoded by thesenucleotide sequences are set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

Nucleic acid molecules that are fragments of the claimed toxin-encodingnucleotide sequences are also encompassed by the present invention. By“fragment” is intended a portion of the nucleotide sequence encoding afragment of a claimed modified Cry1Ca toxin. A fragment of a nucleotidesequence may encode a biologically active portion of a claimed toxinprotein, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below.

BRIEF DESCRIPTION OF THE SEQUENCES

-   -   SEQ ID NO:1 DNA sequence encoding DIG-468    -   SEQ ID NO:2 is the DIG-468 protein sequence    -   SEQ ID NO:3 DNA sequence encoding DIG-483    -   SEQ ID NO:4 is the DIG-483 protein sequence    -   SEQ ID NO:5 DNA sequence encoding DIG-485    -   SEQ ID NO:6 is the DIG-485 protein sequence    -   SEQ ID NO:7 DNA sequence encoding DIG-487    -   SEQ ID NO:8 is the DIG-487 protein sequence    -   SEQ ID NO:9 DNA sequence encoding DIG-462    -   SEQ ID NO:10 is the DIG-462 protein sequence    -   SEQ ID NO:11 DNA sequence encoding DIG-463    -   SEQ ID NO:12 is the DIG-463 protein sequence    -   SEQ ID NO:13 DNA sequence encoding DIG-464    -   SEQ ID NO:14 is the DIG-464 protein sequence    -   SEQ ID NO:15 DNA sequence encoding DIG-465    -   SEQ ID NO:16 is the DIG-465 protein sequence    -   SEQ ID NO:17 DNA sequence encoding DIG-466    -   SEQ ID NO:18 is the DIG-466 protein sequence    -   SEQ ID NO:19 DNA sequence encoding DIG-467    -   SEQ ID NO:20 is the DIG-467 protein sequence    -   SEQ ID NO:21 DNA sequence encoding DIG-469    -   SEQ ID NO:22 is the DIG-469 protein sequence    -   SEQ ID NO:23 DNA sequence encoding DIG-473    -   SEQ ID NO:24 is the DIG-473 protein sequence    -   SEQ ID NO:25 DNA sequence encoding DIG-474    -   SEQ ID NO:26 is the DIG-474 protein sequence    -   SEQ ID NO:27 DNA sequence encoding DIG-482    -   SEQ ID NO:28 is the DIG-482 protein sequence    -   SEQ ID NO:29 DNA sequence encoding modified Cry1Ca codon        optimized for maize (IRDIG544.11)    -   SEQ ID NO:30 is the modified Cry1Ca protein toxin sequence        (IRDIG544.11)    -   SEQ ID NO:31 DNA sequence encoding a modified Cry1Ca,        IRDIG544.12, with high GC codon optimization    -   SEQ ID NO:32 protein toxin sequence for IRDIG544.12    -   SEQ ID NO:33 a dicot optimized DNA sequence encoding modified        Cry1Ca, IRDIG544.9    -   SEQ ID NO:34 protein sequence of modified Cry1Ca, IRDIG544.9    -   SEQ ID NO:35 a dicot optimized DNA sequence encoding modified        Cry1Ca, IRDIG544.8    -   SEQ ID NO:36 IRDIG544.8 protein codon optimized for dicots    -   SEQ ID NO:37 DNA sequence encoding a modified Cry1Ca toxin fused        to the Cry1Ab protoxin segment    -   SEQ ID NO:38 is the protein toxin sequence produced from the DNA        of SEQ ID NO:37    -   SEQ ID NO:39 High GC codon optimized DNA sequence encoding a        modified Cry1Ca, IRDIG544.12, fused to TraP12    -   SEQ ID NO:40 Is the modified Cry1Ca toxin, IRDIG544.12, fused to        TraP12

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows expression levels of DIG-465 by construct 115752 andDIG-473 by construct 115753 in T₁ maize leaves sampled by leaf punches.

FIG. 2 is a plot of the amount of leaf damage in maize caused by FAW orCry1Fa resistant FAW versus the level of expression of DIG-465.

FIG. 3 is a plot of the amount of leaf damage in maize caused by FAW orCry1Fa resistant FAW versus the level of expression of DIG-473.

DETAILED DESCRIPTION OF THE INVENTION

By the use of the term “genetic material” herein, it is meant to includeall genes, nucleic acid, DNA and RNA. These sequences have been alteredin such a manner to increase the stability of the expressed proteintoxin when the gene is transformed in a plant, specifically maize anddicots. The protein toxins discussed herein are typically referred to as“insecticides” or “insecticidal”. By insecticides and insecticidal it ismeant herein that the protein toxins have a “functional activity” asfurther defined herein and are used as insect control agents.

By “functional activity” it is meant herein that the protein toxinsfunction as insect control agents in that the proteins are orallyactive, or have a toxic effect, or are able to disrupt or deter feeding,which may or may not cause death of the insect. When an insect comesinto contact with an effective amount of toxin delivered via transgenicplant expression, formulated protein composition(s), sprayable proteincomposition(s), a bait matrix or other delivery system, the results aretypically death of the insect, or the insects do not feed upon thesource which makes the toxins available to the insects.

By the use of the term “oligonucleotides” it is meant a macromoleculeconsisting of a short chain of nucleotides of either RNA or DNA. Suchlength could be at least one nucleotide, but typically are in the rangeof about 10 to about 12 nucleotides. The determination of the length ofthe oligonucleotide is well within the skill of an artisan and shouldnot be a limitation herein. Therefore, oligonucleotides may be less than10 or greater than 12. The subject invention concerns not only thepolynucleotide sequences which encode these classes of toxins, but alsothe use of these polynucleotide sequences to produce recombinant hostswhich express the toxins.

By the use of the term “toxic” or “toxicity” as used herein it is meantthat the toxins produced by Bacillus thuringiensis have “functionalactivity” as defined herein.

By use of the term “modified Cry1Ca toxin(s)” it is meant to include allof the protein sequences of the Sequence Listing and all the variantsthereof described herein.

By the use of the term “genetic material” herein, it is meant to includeall genes, nucleic acid, DNA and RNA.

For designations of nucleotide residues of polynucleotides, DNA, RNA,oligonucleotides, and primers, and for designations of amino acidresidues of proteins, standard IUPAC abbreviations are employedthroughout this document. Nucleic acid sequences are presented in thestandard 5′ to 3′ direction, and protein sequences are presented in thestandard amino (N) terminal to carboxy (C) terminal direction.

The toxins and genes of the subject invention can be further defined bytheir amino acid and nucleotide sequences, and the sequence of uniquefragments comprised by the full-length DNA and amino sequences. Thesequences of the molecules within each novel class can be defined hereinin terms of homology to certain exemplified sequences as well as interms of the ability to hybridize with, or be amplified by, certainexemplified probes and primers. The classes of toxins provided hereincan also be identified based on their immunoreactivity with certainantibodies.

Toxin structure. The toxin of the subject invention can also becharacterized in terms of the structure and domain composition. Thecorrelation of protein sequence variability with differences inbioactivity spectrum led to early hypotheses that the “hypervariable”regions between blocks three and five are responsible for differences ininsect specificity among B.t. delta-endotoxins.

When the gene that encodes native full length Cry1Ca protein wasinserted and expressed in maize cells, at least 5 detectable proteolyticdegradation products were observed. Those five polypeptides weredetermined to have the following amino acid lengths: 1-1164, 1-628,29-628, 74-628, and 74-596. Of the five Cry1Ca degradation productsdetected, two of the fragments were found to be inactive against the keydriver insect pests. In most cases, these two inactive fragmentsrepresented a major portion of the Cry1Ca-related proteins detected inmaize cells. Expressing the native, full length gene for Cry1Ca in maizeresulted in plants having insufficient functional activity against keyinsect pests such as S. frugiperda.

When the gene that expresses truncated native Cry1Ca protein (aa 1-628)was inserted and expressed in maize cells, less proteolytic processingoccurred. The majority remained unprocessed and functionally active.Thus expressing the truncated Cry1Ca gene in maize cells resulted inplants having sufficient functional activity against key insect pestsdue to reduced proteolysis in maize cells.

Altering the primary amino acid sequence of Cry1Ca allows for continualbiological activity against key insect pests, and results in lessproteolytic processing of the protein, as measured in vitro usingchymotrypsin as the protease enzyme. Less proteolytic processing ofaltered Cry1Ca protein results in higher amounts of functionally activeprotein accumulating in plants and results in greater activity againstthe target insect pests.

Protease sensitive variants. Insect gut proteases typically function inaiding the insect in obtaining needed amino acids from dietary protein.The best understood insect digestive proteases are serine proteases,which appear to be the most common type (Englemann and Geraerts, 1980),particularly in Lepidopteran species. Coleopteran insects have guts thatare more neutral to acidic than are Lepidopteran guts. The majority ofColeopteran larvae and adults, for example Colorado potato beetle, haveslightly acidic midguts, and cysteine proteases provide the majorproteolytic activity (Wolfson and Murdock, 1990). More precisely, Thieand Houseman (1990) identified and characterized the cysteine proteases,cathepsin B-like and cathepsin H-like, and the aspartyl protease,cathepsin D-like, in Colorado potato beetle. Gillikin et al., (1992)characterized the proteolytic activity in the guts of western cornrootworm larvae and found primarily cysteine proteases. U.S. Pat. No.7,230,167 disclosed that the serine protease, cathepsin G, exists inwestern corn rootworm. The diversity and different activity levels ofthe insect gut proteases may influence an insect's sensitivity to aparticular B.t. toxin.

In one embodiment, the toxins have specific changes in their amino acidsequences that significantly reduce the level of protease processing ofthe expressed protein by proteases found naturally in maize plants. Thechanges in amino acids results in higher levels of functional activityof the protein when expressed in maize. Protease cleavage sites may beintroduced at desired locations by chemical gene synthesis or spliceoverlap PCR (Horton et al., 1989). Serine protease recognitionsequences, for example, can optionally be inserted at specific sites inthe Cry protein structure to affect protein processing at desireddeletion points within the midgut of certain insect pests. Lepidopteranmidgut serine proteases such as trypsin or trypsin-like enzymes,chymotrypsin, elastase, etc. (Christeller et al., 1992) can be exploitedfor activation of Cry proteins by engineering protease recognitionsequences at desired processing sites. Likewise, Coleopteran serineproteases such as trypsin, chymotrypsin and cathepsin G-like proteasemay similarly be exploited by engineering recognition sequences atdesired processing sites. Further, Coleopteran cysteine proteases suchas cathepsins (B-like, L-like, O-like, and K-like proteases) (Koiwa etal., 2000 and Bown et al., 2004), metalloproteases such as ADAM10(Ochoa-Campuzano et al., 2007), and aspartic acid proteases such ascathepsins D-like and E-like, pepsin, plasmepsin, and chymosin may beexploited by engineering recognition sequences at desired processingsites.

The scope of this invention includes variant Cry1Ca insecticidalproteins that are produced by manipulating the encoding sequence for thesubject insecticidal proteins by introduction or elimination of proteaseprocessing sites at appropriate positions to allow, or eliminate,proteolytic cleavage of a larger variant protein by insect, plant, ormicroorganism proteases. The end result of such manipulation is thegeneration of toxin molecules having the same or better activity as theintact (full length) native toxin protein.

Unlike the high sequence specificity associated with Type II restrictionendonucleases in the recognition and cleavage of their DNA substrates,proteolytic enzymes are more nonspecific in the amino sequencecomprising the cleavage recognition site. Some generalities have beendiscovered regarding the amino acid structures comprising some proteasecleavage sites, in particular, cathepsin G as compared to cathepsins B,K, L, and S (Bown et al., 2004). In the nomenclature of proteasecleavage sites in the illustrations below, the amino acid residuesupstream (i.e. towards the N-terminus) from the cleavage site arenumbered P1, P2, P3, P4, P5, etc, with residue P1 being immediatelyadjacent to the cleavage site, and residue P5 being the fifth mostdistal from the cleavage site in the N-terminal direction. Amino acidresidues downstream (i.e. towards the C-terminus) from the cleavage siteare numbered P1′, P2′, P3′, P4′, P5′ etc., with residue P1′ beingimmediately adjacent to the cleavage site, and residue P5′ being thefifth most distal from the cleavage site in the C-terminal direction.Cathepsin G is known to exhibit preferential cleavage after P1 residuesglutamine, lysine, tryptophan, or phenylalanine, where residues P2, P3,P4, P5, etc., and P1′, P2′, P3′, P4′ P5′, etc. can be any of the 20amino acids normally found in natural proteins. Somewhat enhancedcleavage site sequence specificity is demonstrated by cathepsins B, K,L, and S, wherein the side chain of the P2 amino acid fits into asubstrate binding site S2 of the cathepsin. The S2 site of thesecathepsins preferentially interacts with P2 amino acids having largehydrophobic side chains (e.g. as found in valine, leucine, isoleucine,phenylalanine, tryptophan, and tyrosine), and disfavors interaction withP2 residues having charged side chains (except that cathepsins B and Laccept the large hydrophilic charged side chain of arginine in the P2position). Some specificity is seen in the identity of the amino acid inthe P3 position. For example, cathepsin L cleaves preferentially afterarginine in the P1 position, when phenylalanine or arginine occupy theP2 position. The P3 amino acid can be either an aromatic type (e.g.phenylalanine, tryptophan, histidine, or tyrosine) or a hydrophobic type(e.g. alanine, valine, leucine, isoleucine, phenylalanine, tryptophan,or tyrosine). Positions P4, P5, etc. and P1′, P2′, P3′, P4′, P5′, etc.can be any of the 20 amino acids normally found in natural proteins.

Proteolytic cleavage is further dependent on the availability of thesubject cleavage sequence to the respective protease; sequestration ofthe potential cleavage site within the three-dimensional structure ofthe protein may render the protein resistant to cleavage by theparticular protease. It is thought that the diversity and differentactivity levels of the insect gut proteases may influence an insect'ssensitivity to a particular B.t. toxin. One skilled in the arts ofbiochemistry and molecular biology can examine the biochemicalcharacteristics (including, but not limited to, determination of thesequences of the amino acids comprising the N-terminus and C-terminus ofthe polypeptide) of insecticidal protein fragments generated by proteasecleavage/activation of larger proteins by the gut proteases ofsusceptible insects. One may also characterize the protease regime ofthe guts of nonsusceptible insects or host plants, and engineer, atappropriate places within the coding sequence for the B.t. insecticidalprotein, sequences amenable to cleavage by the gut proteases ofnonsusceptible insects or prospective host plants in which the B.t.insecticidal protein will be produced transgenically. Such analyses andmanipulations of the subject B.t. insecticidal protein are understood tobe within the scope of this invention.

In another embodiment, the toxins have specific changes in their aminoacid sequences that significantly enhance the level of proteinexpression, when expressed in a variety of different expression systems,including plant and bacteria. The result of the increased expression ofthe protein is increased functional activity in the expression system.This is advantageous in providing a high dose of the toxin to the insectwhich can prevent the occurrence of resistance in the insects to thetoxins due to survival of small populations of insects receiving a sublethal dose of the protein toxin.

Genes and toxins. The protein molecules of the embodiments hereincomprise amino acid sequences that are homologous to known pesticidalproteins, particularly B.t. Cry proteins, more particularly Cry1Caprotein (Genbank Accession No. AAA22343). The predicted amino acidsequences encoded by a nucleotide sequences of the embodiments aredisclosed as SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, and 40.

The sequence of toxins of the subject invention are provided as SEQ IDNOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, and 40. In a preferred embodiment, the toxins of the subjectinvention have at least one of the following characteristics:

-   -   (a) said toxin is encoded by a nucleotide sequence which        hybridizes under stringent conditions with a nucleotide sequence        selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,        11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or        their complementary sequences.    -   (b) said toxin is immunoreactive with an antibody raised against        an approximately 68-71 kDa pesticidal toxin, or a fragment        thereof, from a Bacillus thuringiensis isolate.    -   (c) said toxin is encoded by a nucleotide sequence wherein a        portion of said nucleotide sequence can be amplified by PCR        using a primer pair to produce a fragment of about 25-40 bp,    -   (d) said toxin comprises a pesticidal portion of the amino acid        sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18,        20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40,    -   (e) said toxin comprises an amino acid sequence which has at        least about (90%) homology with a pesticidal portion of an amino        acid sequence selected from the group consisting of SEQ ID        NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,        34, 36, 38, and 40,    -   (f) said toxin is encoded by a nucleotide sequence which        hybridizes under stringent conditions with an insecticidal        portion of a nucleotide sequence selected from the group        consisting of DNA which encodes SEQ ID NOs:2, 4, 6, 8, 10, 12,        14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40,    -   (g) said toxin is immunoreactive with an antibody to an        approximately 68 kDa or 130 kDa pesticidal toxin, or a fragment        thereof, from a Bacillus thuringiensis isolate, MR-1206.    -   (h) said toxin comprises an amino acid sequence which has at        least about (90%) homology with an amino acid sequence selected        from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14,        16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40 and        pesticidal portions of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16,        18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

The specific genes exemplified herein, variations of these genes, andfragments of these genes may also be obtained, for example, by syntheticconstruction by methods currently practiced by any of several commercialsuppliers (see for example, U.S. Pat. No. 7,482,119). These genes, orportions or variants thereof, may also be constructed synthetically, forexample, by use of a gene synthesizer and the methods of, for example,U.S. Pat. No. 5,380,831. Alternatively, variations of synthetic ornaturally occurring genes may be readily constructed using standardmolecular biological techniques for making point mutations. Fragments ofthese genes can also be made using commercially available exonucleasesor endonucleases according to standard procedures. For example, enzymessuch as Bal31 or site-directed mutagenesis can be used to systematicallycut off nucleotides from the ends of these genes. Also, gene fragmentswhich encode active toxin fragments may be obtained using a variety ofrestriction enzymes.

Nucleic acid molecules that are fragments of a claimed toxin-encodingnucleotide sequence comprise at least about 15, 20, 30, 40, 50, 60, 75,100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 3000, 3500 nucleotides, or up to thenumber of nucleotides present in a full-length claimed insecticidaltoxin-encoding nucleotide sequence disclosed herein (for example, 1,878nucleotides for SEQ ID NO:1; 3,495 nucleotides for SEQ ID NO:37),depending upon the intended use. A fragment of a nucleotide sequencethat encodes a biologically active portion of a claimed protein of theinvention will encode at least about 15, 25, 30, 40, 50, 75, 100, 125,150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800,900, 1000, 1100 or 1200 contiguous amino acids, or up to the totalnumber of amino acids present in a full-length insecticidal protein ofthe invention (for example, 625 amino acids for SEQ ID NO:2 or 1,164amino acids for SEQ ID NO:38).

Recombinant hosts. The toxin-encoding genes of the subject invention canbe introduced into a wide variety of microbial or plant hosts.Expression of the toxin gene results, directly or indirectly, in theintracellular production and maintenance of the pesticidal protein. Withsuitable microbial hosts, e.g. Pseudomonas, the microbes can be appliedto the environment of the pest, where they will proliferate and can beingested. The result is control of the pest. Alternatively, the microbehosting the toxin gene can be treated under conditions that prolong theactivity of the toxin and stabilize the cell. The treated cell, whichretains the toxic activity, then can be applied to the environment ofthe target pest.

Where the toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type indigenous microorganisms,provide for stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environmental degradation and inactivation.

B.t. spores or recombinant host cells also can be treated prior to beingapplied or formulated for application to plants. For example, isolatedB.t. spores and/or toxin crystals can be chemically treated to prolonginsecticidal activity and thereby include a treated polypeptide of theinvention (U.S. Pat. No. 4,695,462 and Gaertner et al., 1993).

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g. genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Sinorhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium,Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, andAlcaligenes; and fungi, particularly yeast, e.g. genera Saccharomyces,Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, andAureobasidium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacterium tumefaciens,Agrobacterium radiobacter, Rhodopseudomonas spheroides, Xanthomonascampestris, Sinorhizobium meliloti (formerly Rhizobium meliloti),Alcaligenes eutrophus, and Azotobacter vinelandii; and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms.

A preferred embodiment of the subject invention is the transformation ofplants with genes encoding the subject insecticidal protein or itsvariants. The transformed plants are resistant to attack by an insecttarget pest by virtue of the presence of controlling amounts of thesubject insecticidal protein or its variants in the cells of thetransformed plant. By incorporating genetic material that encodes theinsecticidal properties of the B.t. insecticidal toxins into the genomeof a plant eaten by a particular insect pest, the adult or larvae woulddie after consuming the food plant. Numerous members of themonocotyledonous and dicotyledonous classifications have beentransformed. Transgenic agronomic crops as well as fruits and vegetablesare of commercial interest. Such crops include but are not limited tomaize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat,cotton, peanuts, tomatoes, potatoes, and the like. Several techniquesexist for introducing foreign genetic material into monocot or dicotplant cells, and for obtaining fertile plants that stably maintain andexpress the introduced gene. Such techniques include acceleration ofgenetic material coated onto microparticles directly into cells (U.S.Pat. Nos. 4,945,050 and 5,141,131). Plants may be transformed usingAgrobacterium technology, see U.S. Pat. Nos. 5,177,010, 5,104,310,European Patent Application 0131624B1, European Patent Application120516, European Patent Application 159418B, European Patent Application176112, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763, 4,940,838,4,693,976, European Patent Application 116718, European PatentApplication 290799, European Patent Application 320500, European PatentApplication 604662, European Patent Application 627752, European PatentApplication 0267159, European Patent Application 0292435, U.S. Pat. Nos.5,231,019, 5,463,174, 4,762,785, 5,004,863, and 5,159,135. Othertransformation technology includes WHISKERS™ technology, see U.S. Pat.Nos. 5,302,523 and 5,464,765. Electroporation technology has also beenused to transform plants, see WO 87/06614, U.S. Pat. Nos. 5,472,869,5,384,253, WO9209696, and WO9321335. All of these transformation patentsand publications are incorporated by reference. In addition to numeroustechnologies for transforming plants, the type of tissue which iscontacted with the foreign genes may vary as well. Such tissue wouldinclude but would not be limited to embryogenic tissue, callus tissuetype I and II, hypocotyl, meristem, and the like. Almost all planttissues may be transformed during dedifferentiation using appropriatetechniques within the skill of an artisan.

Genes encoding modified Cry1Ca insecticidal toxins and variants can beinserted into plant cells using a variety of techniques which are wellknown in the art as disclosed above. For example, a large number ofcloning vectors comprising a marker that permits selection of thetransformed microbial cells and a replication system functional in E.co/i are available for preparation and modification of foreign genes forinsertion into higher plants. Such manipulations may include, forexample, the insertion of mutations, truncations, additions, deletions,or substitutions as desired for the intended use. The vectors comprise,for example, pBR322, pUC series, M13mp series, pACYC184, etc.Accordingly, the sequence encoding the Cry protein or variants can beinserted into the vector at a suitable restriction site. The resultingplasmid is used for transformation of E. coli, the cells of which arecultivated in a suitable nutrient medium, then harvested and lysed sothat workable quantities of the plasmid are recovered. Sequenceanalysis, restriction fragment analysis, electrophoresis, and otherbiochemical-molecular biological methods are generally carried out asmethods of analysis. After each manipulation, the DNA sequence used canbe cleaved and joined to the next DNA sequence. Each manipulated DNAsequence can be cloned in the same or other plasmids.

Depending on the plant transformation method, ancillary DNA sequencesmay be necessary. If, for example, a Ti or Ri plasmid is used for thetransformation of the plant cell, then at least a T-DNA right borderrepeat, but often both the right border repeat and the left borderrepeat of the Ti or Ri plasmid will be joined as the flanking region ofthe genes desired to be inserted into the plant cell. The use ofT-DNA-containing vectors for the transformation of plant cells has beenintensively researched and sufficiently described in EP 120516; Lee andGelvin (2008), Fraley et al., (1986), and An et al., (1985), and is wellestablished in the field.

Once the inserted DNA has been integrated into the plant genome, it isrelatively stable throughout subsequent generations. The vector used totransform the plant cell normally contains a selectable marker geneencoding a protein that confers on the transformed plant cells toleranceto a herbicide or an antibiotic, such as bialaphos, kanamycin, G418,bleomycin, or hygromycin, inter alia. The individually employedselectable marker gene should accordingly permit the selection oftransformed cells while the growth of cells that do not contain theinserted DNA is suppressed by the selective compound.

A large number of techniques are available for inserting DNA into a hostplant cell. Those techniques include transformation with T-DNA deliveredby Agrobacterium tumefaciens or Agrobacterium rhizogenes as thetransformation agent. Additionally, fusion of plant protoplasts withliposomes containing the DNA to be delivered, direct injection of theDNA, biolistics transformation (microparticle bombardment), orelectroporation, as well as other possible methods, may be employed. Oneskilled in the field of plant transformation will understand thatmultiple methodologies are available for the production of transformedplants, and they may be modified and specialized to accommodatebiological differences between various host plant species.

If Agrobacterium strains are used for the transformation, the DNA to beinserted will be cloned into special plasmids, namely either into anintermediate (shuttle) vector or into a binary vector. The intermediatevectors can be integrated into the Ti or Ri plasmid or derivativesthereof by homologous recombination owing to sequences that arehomologous between the Ti or Ri plasmid and the intermediate plasmid.The Ti or Ri plasmid also comprises the vir region comprising vir genesnecessary for the transfer of the T-DNA. Intermediate vectors cannotreplicate in Agrobacteria. The intermediate vector can be transferredinto Agrobacterium tumefaciens by means of a helper plasmid (viabacterial conjugation), by electroporation, by direct DNA, chemicallymediated transformation, or by other methodologies. Binary vectors canreplicate autonomously in both E. coli and Agrobacterium cells. Theycomprise sequences, framed by the right and left T-DNA border repeatregions, that may include a selectable marker gene functional for theselection of transformed plant cells, a cloning linker, cloningpolylinker, or other sequence which can function as an introduction sitefor genes destined for plant cell transformation. They can betransformed directly into Agrobacterium cells (Holsters et al., (1978))by electroporation, or by direct DNA, chemically mediatedtransformation, or introduced by bacterial conjugation, or by othermethodologies. The Agrobacterium used as host cell is to comprise aplasmid carrying a vir region. The vir region is necessary for thetransfer of the T-DNA into the plant cell. Additional T-DNA regionsadditive to the one containing the gene encoding the B.t. insecticidaltoxin protein or its variants may be present in the Agrobacterium hostcell. The bacterium cells so transformed are used for the transformationof plant cells. Plant explants (for example, pieces of leaf, segments ofstalk, roots, but also protoplasts or suspension-cultivated cells) canadvantageously be cultivated with Agrobacterium tumefaciens orAgrobacterium rhizogenes for the transfer of the DNA into the plantcell. Whole plants may then be regenerated from the infected plantmaterial following placement in suitable growth conditions and culturemedium, which may contain antibiotics or herbicides for selection of thetransformed plant cells. The plants so obtained can then be tested forthe presence of the inserted DNA.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants. Such plants can be grown in the normal manner and crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties, for example, the ability to controlthe feeding of plant pest insects.

No special demands are made in the construction of the plasmids in thecase of those used for injection and electroporation. It is possible touse ordinary plasmids, such as, for example, pUC derivativesappropriately modified to contain all the genes desired to betransferred to plant cells.

The activity of recombinant polynucleotides inserted into plant cellscan be dependent upon the influence of endogenous plant DNA adjacent tothe insert. Thus, another option is to take advantage of events that areknown to be excellent locations in a plant genome for insertions. Seee.g. WO 2005/103266 A1, relating to Cry1F and Cry1Ac cotton events; thesubject B.t. insecticidal toxin gene can be substituted in those genomicloci in place of the Cry1F or Cry1Ac inserts. Targeted homologousrecombination, for example, can be used according to the subjectinvention. This type of technology is the subject of, for example, WO03/080809 and the corresponding published U.S. application (USPA20030232410), relating to the use of zinc fingers for targetedrecombination. The use of recombinases (cre-lox and flp-frt for example)is also known in the art.

In a preferred embodiment of the subject invention, plants will betransformed with genes wherein the codon usage of the protein codingregion has been optimized for plants. See, for example, U.S. Pat. No.5,380,831, which is hereby incorporated by reference. Also,advantageously, plants encoding a truncated toxin will be used. Thetruncated toxin typically will encode about 55% to about 80% of the fulllength toxin. Methods for creating synthetic B.t. genes for use inplants are known in the art (Stewart 2007).

Another variable is the choice of a selectable marker. The preferencefor a particular marker is at the discretion of the artisan, but any ofthe following selectable markers may be used along with any other genenot listed herein which could function as a selectable marker. Suchselectable markers include but are not limited to aminoglycosidephosphotransferase gene of transposon Tn5 (Aph II) which encodesresistance to the antibiotics kanamycin, neomycin and G418, as well asthose genes which code for tolerance to glyphosate; hygromycin;methotrexate; phosphinothricin (bialaphos); imidazolinones,sulfonylureas and triazolopyrimidine herbicides, such as chlorosulfuron;bromoxynil, dalapon and the like. Examples of such genes are provided inMerlo, (2002), which is incorporated herein by reference.

In addition to a selectable marker, it may be desirable to use areporter gene. In some instances a reporter gene may be used without aselectable marker. Reporter genes are genes which typically do notprovide a growth advantage to the recipient organism or tissue. Thereporter gene typically encodes for a protein which provides for somephenotypic change or enzymatic property. A preferred reporter gene isthe glucuronidase (GUS) gene. Other examples of reporter genes areprovided in Merlo (2002).

Regardless of transformation technique, the gene is preferablyincorporated into a gene transfer vector adapted to express the B.tinsecticidal toxin genes and variants in the plant cell by including inthe vector a plant promoter. In addition to plant promoters, promotersfrom a variety of sources can be used efficiently in plant cells toexpress foreign genes. For example, promoters of bacterial origin, suchas the octopine synthase promoter, the nopaline synthase promoter, themannopine synthase promoter; promoters of viral origin, such as the 35Sand 19S promoters of cauliflower mosaic virus, and the like may be used.Plant promoters include, but are not limited toribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),beta-conglycinin promoter, phaseolin promoter, ADH (alcoholdehydrogenase) promoter, heat-shock promoters, ADF (actindepolymerization factor) promoter, and tissue specific promoters.Promoters may also contain certain enhancer sequence elements that mayimprove the transcription efficiency. Typical enhancers include but arenot limited to ADH1-intron 1 and ADH1-intron 6. Constitutive promotersmay be used. Constitutive promoters direct continuous gene expression innearly all cells types and at nearly all times (e.g., actin, ubiquitin,CaMV 35S). Tissue specific promoters are responsible for gene expressionin specific cell or tissue types, such as the leaves or seeds (e.g.,zein, oleosin, napin, ACP (Acyl Carrier Protein)), and these promotersmay also be used. Promoters may also be used that are active during acertain stage of the plants' development as well as active in specificplant tissues and organs. Examples of such promoters include but are notlimited to promoters that are root specific, pollen-specific, embryospecific, corn silk specific, cotton fiber specific, seed endospermspecific, phloem specific, and the like.

Under certain circumstances it may be desirable to use an induciblepromoter. An inducible promoter is responsible for expression of genesin response to a specific signal, such as: physical stimulus (e.g. heatshock genes); light (e.g. RUBP carboxylase); hormone (e.g.glucocorticoid); antibiotic (e.g. tetracycline); metabolites; and stress(e.g. drought). Other desirable transcription and translation elementsthat function in plants may be used, such as 5′ untranslated leadersequences, RNA transcription termination sequences and poly-adenylateaddition signal sequences. Numerous plant-specific gene transfer vectorsare known in the art.

The subject invention includes plant cells that are not totipotent(non-totipotent), plant cells that are not propagative material (forexample, leaf cells in some embodiments; seed cells are excluded fromsome embodiments) and are incapable of differentiating into wholeplants. The subject invention includes plant cells that have uses otherthan for regenerating into a whole plant. For example, said plant cellscan be used to produce a protein (such as a DIG-465 protein of thesubject invention). Thus, plant cells of the subject invention includethose that have uses other than totipotency (that is, some cells ofsubject invention are not regenerable into a whole plant). However, someembodiments do include seed cells and plant cells that can beregenerated into a whole plant.

A further method for identifying the toxins and genes of the subjectinvention is through the use of oligonucleotide probes. These probes aredetectable nucleotide sequences. These sequences may be rendereddetectable by virtue of an appropriate radioactive label or may be madeinherently fluorescent as described in U.S. Pat. No. 6,268,132. As iswell known in the art, if the probe molecule and nucleic acid samplehybridize by forming strong base-pairing bonds between the twomolecules, it can be reasonably assumed that the probe and sample havesubstantial sequence homology. Preferably, hybridization is conductedunder stringent conditions by techniques well-known in the art, asdescribed, for example, in Keller and Manak (1993). Detection of theprobe provides a means for determining in a known manner whetherhybridization has occurred. Such a probe analysis provides a rapidmethod for identifying toxin-encoding genes of the subject invention.The nucleotide segments which are used as probes according to theinvention can be synthesized using a DNA synthesizer and standardprocedures. These nucleotide sequences can also be used as PCR primersto amplify genes of the subject invention.

As used herein the terms “stringent conditions” or “stringenthybridization conditions” are intended to refer to conditions underwhich a probe will hybridize (anneal) to its target sequence to adetectably greater degree than to other sequences (e.g. at least 2-foldover background). Stringent conditions are sequence-dependent and willbe different in different circumstances. By controlling the stringencyof the hybridization and/or washing conditions, target sequences thatare 100% complementary to the probe can be identified (homologousprobing). Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to pH 8.3 and thetemperature is at least about 30° C. for short probes (e.g. 10 to 50nucleotides) and at least about 60° C. for long probes (e.g. greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C. and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate)at 50° C. to 55° C. Exemplary moderate stringency conditions includehybridization in 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C. anda wash in 0.5× to 1×SSC at 55° C. to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C. and a wash in 0.1×SSC at 60° C. to 65° C. Optionally, washbuffers may comprise about 0.1% to about 1% SDS. Duration ofhybridization is generally less than about 24 hours, usually about 4 toabout 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA/DNA hybrids, the thermal melting point (Tm) isthe temperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm,hybridization conditions, and/or wash conditions can be adjusted tofacilitate annealing of sequences of the desired identity. For example,if sequences with >90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the Tm for the specific sequence and its complement at a definedionic strength and pH. However, highly stringent conditions can utilizea hybridization and/or wash at 1° C., 2° C., 3° C., or 4° C. lower thanthe Tm; moderately stringent conditions can utilize a hybridizationand/or wash at 6° C., 7° C., 8° C., 9° C., or 10° C. lower than the Tm,and low stringency conditions can utilize a hybridization and/or wash at11° C., 12° C., 13° C., 14° C., 15° C., or 20° C. lower than the Tm.

Tm (in ° C.) may be experimentally determined or may be approximated bycalculation. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl (1984):

Tm(° C.)=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% formamide)−500/L;

where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % formamide is thepercentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs

Alternatively, the Tm is described by the following formula (Beltz etal., 1983).

Tm(° C.)=81.5° C.+16.6(log[Na+])+0.41(% GC)−0.61(% formamide)−600/L

where [Na+] is the molarity of sodium ions, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % formamide is thepercentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs

Using the equations, hybridization and wash compositions, and desiredTm, those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a Tm of lessthan 45° C. (aqueous solution) or 32° C. (formamide solution), it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993) and Ausubel et al., (1995). Also see Sambrook etal., (1989).

Hybridization of immobilized DNA on Southern blots with radioactivelylabeled gene-specific probes may be performed by standard methods(Sambrook et al., supra). Radioactive isotopes used for labelingpolynucleotide probes may include 32P, 33P, 14C, or 3H. Incorporation ofradioactive isotopes into polynucleotide probe molecules may be done byany of several methods well known to those skilled in the field ofmolecular biology. (See, e.g. Sambrook et al., supra.) In general,hybridization and subsequent washes may be carried out under stringentconditions that allow for detection of target sequences with homology tothe claimed toxin encoding genes. For double-stranded DNA gene probes,hybridization may be carried out overnight at 20-25° C. below the Tm ofthe DNA hybrid in 6×SSPE, 5×Denhardt's Solution, 0.1% SDS, 0.1 mg/mLdenatured DNA [20×SSPE is 3M NaCl, 0.2 M NaHPO4, and 0.02M EDTA(ethylenediamine tetra-acetic acid sodium salt); 100×Denhardt's Solutionis 20 gm/L Polyvinylpyrollidone, 20 gm/L Ficoll type 400 and 20 gm/LBovine Serum Albumin (fraction V)].

Washes may typically be carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash).(2) Once at Tm −20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

For oligonucleotide probes, hybridization may be carried out overnightat 10-20° C. below the Tm of the hybrid in 6×SSPE, 5×Denhardt'ssolution, 0.1% SDS, 0.1 mg/mL denatured DNA. Tm for oligonucleotideprobes may be determined by the following formula (Suggs et al., 1981).

Tm(° C.)=2(number of T/A base pairs)+4(number of G/C base pairs)

Washes may typically be carried out as follows:

(1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (lowstringency wash).(2) Once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1%SDS (moderate stringency wash).

A practitioner skilled in the art will realize that probe molecules forhybridization and hybrid molecules formed between probe and targetmolecules may be rendered detectable by means other than radioactivelabeling.

Variant toxins. The genes and toxins useful according to the subjectinvention include not only the truncated sequences disclosed but alsofull length sequences, fragments of these sequences, variants, mutants,and fusion proteins which retain the characteristic pesticidal activityof the toxins specifically exemplified herein. As used herein, the terms“variants” or “variations” of genes refer to nucleotide sequences whichencode the same toxins or which encode equivalent toxins havingpesticidal activity. Further as used herein, the term “equivalenttoxins” refers to toxins having the same or essentially the samebiological activity against the target pests as the claimed toxins.Thus, the variant or variations of the claimed toxins will have at leastabout 30%, preferably at least about 50%, more preferably at least about70%, even more preferably at least about 80% of the activity of theclaimed toxins. Methods for measuring pesticidal activity are well knownin the art and are exemplified herein. By “variants” it is intendedherein to include proteins or polypeptides having an amino acid sequencethat is at least about 60%, 65%, preferably about 70%, 75%, morepreferably about 80%, 85%, most preferably about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, and 40. Variants also include polypeptides encoded by anucleic acid molecule that hybridizes to the nucleic acid molecule ofSEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, or 39, or a complement thereof, under stringent conditions.Such variants generally will retain the claimed activity. Variantsinclude polypeptides that differ in amino acid sequence due tomutagenesis. Variant proteins encompassed by the present invention areinsecticidally active.

Variant proteins can also be designed that differ at the primary aminoacid sequence level and which retain the same or similar overallessential three-dimensional structure, surface charge distribution, andthe like. See e.g. U.S. Pat. No. 7,058,515; Larson et al., (2002);Crameri et al., (1997); Stemmer, W. P. C. (1994a); Stemmer, W. P. C.(1994b)” Stemmer, W. P. C. (1995); Crameri et al., (1996a); and Crameriet al., (1996b).

Certain toxins of the subject invention have been specificallyexemplified herein. Since these toxins are merely exemplary of thetoxins of the subject invention, it should be readily apparent that thesubject invention comprises variant or equivalent toxins (and nucleotidesequences coding for equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave amino acid homology with an exemplified toxin. The amino acididentity will typically be greater than 60%, preferably be greater than75%, more preferably greater than 80%, more preferably greater than 90%,and can be greater than 95%. The amino acid homology will be highest incritical regions of the toxin which account for biological activity orare involved in the determination of three-dimensional configurationwhich ultimately is responsible for the biological activity. In thisregard, certain amino acid substitutions are acceptable and can beexpected if these substitutions are in regions which are not critical toactivity or are conservative amino acid substitutions which do notaffect the three-dimensional configuration of the molecule. For example,amino acids may be placed in the following classes: non-polar, unchargedpolar, basic, and acidic. Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the subject invention so long as thesubstitution does not materially alter the biological activity of thecompound. Table 1 provides a listing of examples of amino acidsbelonging to each class.

TABLE 1 Classes of Amino Acids and Examples. Class of Amino AcidExamples of Amino Acids Nonpolar Side Chains Ala, Val, Leu, Ile, Pro,Met, Phe, Trp Uncharged Polar Side Chains Gly, Ser, Thr, Cys, Tyr, Asn,Gln Acidic Side Chains Asp, Glu Basic Side Chains Lys, Arg, HisBeta-branched Side Chains Thr, Val, Ile Aromatic Side Chains Tyr, Phe,Trp, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin.

Preferred insecticidal toxins proteins of the present invention areencoded by a nucleotide sequence sufficiently identical to thenucleotide sequences of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37 or 39. By “sufficiently identical” isintended an amino acid or nucleotide sequence that has at least about60% or 65% sequence identity, preferably about 70% or 75% sequenceidentity, more preferably about 80% or 85% sequence identity, mostpreferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity compared to a reference sequence as analyzed by one ofthe alignment programs described herein, employing standard parameters.One of skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.percent identity=number of identical positions/total number of positions(e.g. overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990), modified as in Karlin andAltschul (1993). Such an algorithm is incorporated into the BLASTN andBLASTX programs of Altschul et al., (1990). BLAST searches may beconveniently used to identify sequences homologous (similar) to a querysequence in nucleic or protein databases. BLAST nucleotide searches canbe performed with the BLASTN program, score=100, word length=12, toidentify nucleotide sequences having homology to claimed nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe BLASTX program, score=50, word length=3, to identify amino acidsequences having homology to claimed insecticidal protein molecules ofthe invention.

To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997). Alternatively,PSI-Blast can be used to perform an iterated search that detects distantrelationships between molecules Altschul et al., (1997). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g. BLASTX and BLASTN) can be used. Seewww.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

A non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the ClustalW algorithm (Thompson et al.,(1994). ClustalW compares sequences and aligns the entirety of the aminoacid or DNA sequence, and thus can provide data about the sequenceconservation of the entire amino acid sequence or nucleotide sequence.The ClustalW algorithm is used in several commercially availableDNA/amino acid analysis software packages, such as the ALIGNX module ofthe Vector NTI Program Suite (Invitrogen, Inc., Carlsbad, Calif.). Whenaligning amino acid sequences with ALIGNX, one may conveniently use thedefault settings with a Gap open penalty of 10, a Gap extend penalty of0.1 and the blosum63mt2 comparison matrix. After aligning two proteinsequences with ALIGNX, the percent amino acid similarity (consensus) oridentity between the two sequences can be assessed. When aligning twoDNA sequences with ALIGNX, one may conveniently use the default settingswith a Gap open penalty of 15, a Gap extend penalty of 6.6 and theswgapdnamt comparison matrix. After aligning two DNA sequences withALIGNX, the percent identity between the two sequences can be assessed.

A second non-limiting example of a software program useful for analysisof ClustalW alignments is GeneDoc™ (developed by Karl Nicholas,http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html).GeneDoc™ allows assessment of amino acid (or DNA) similarity andidentity between multiple proteins.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller (1988).Such an algorithm is incorporated into the wSTRETCHER program, which ispart of the wEMBOSS sequence alignment software package (available athttp://emboss.sourceforge.net/). STRETCHER calculates an optimal globalalignment of two sequences using a modification of the classic dynamicprogramming algorithm which uses linear space. The output is a standardalignment file. The substitution matrix, gap insertion penalty and gapextension penalties used to calculate the alignment may be specified.When utilizing the STRETCHER program for comparing nucleotide sequences,a Gap open penalty of 16 and a Gap extend penalty of 4 can be used. Thescoring matrix file for comparing DNA sequences is EDNAFULL. When usedfor comparing amino acid sequences, a Gap open penalty of 12 and a Gapextend penalty of 2 can be used. The scoring matrix file for comparingprotein sequences is EBLOSUM62.

A further non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Needleman and Wunsch(1970), which is incorporated in the sequence alignment softwarepackages GAP Version 10 and wNEEDLE (http://emboss.sourceforge.net/).GAP version 10 may be used to determine sequence identity or similarityusing the following parameters: for a nucleotide sequence, % identityand % similarity are found using GAP Weight of 50 and Length Weight of3, and the nwsgapdna. cmp scoring matrix. For amino acid sequencecomparison, % identity or % similarity fare determined using GAP weightof 8 and length weight of 2, and the BLOSUM62 scoring program. wNEEDLEreads two input sequences, finds the optimum alignment (including gaps)along their entire length, and writes their optimal global sequencealignment to file. The algorithm uses a dynamic programming method toensure the alignment is optimum, by exploring all possible alignmentsand choosing the best. A scoring matrix is read that contains values forevery possible residue or nucleotide match. wNEEDLE finds the alignmentwith the maximum possible score where the score of an alignment is equalto the sum of the matches taken from the scoring matrix, minus penaltiesarising from opening and extending gaps in the aligned sequences. Thesubstitution matrix and gap opening and extension penalties areuser-specified. When amino acid sequences are compared, a default Gapopen penalty of 10, a Gap extend penalty of 0.5, and the EBLOSUM62comparison matrix are used. When DNA sequences are compared usingwNEEDLE, a Gap open penalty of 10, a Gap extend penalty of 0.5, and theEDNAFULL comparison matrix are used.

Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by ALIGNX, wNEEDLE, or wSTRETCHER. The% identity is the percentage of identical matches between the twosequences over the reported aligned region (including any gaps in thelength) and the % similarity is the percentage of matches between thetwo sequences over the reported aligned region (including any gaps inthe length).

Toxin fragments and equivalents. Fragments and equivalents which retainthe pesticidal activity of the exemplified toxins would be within thescope of the subject invention. Also, because of the redundancy of thegenetic code, a variety of different DNA sequences can encode the aminoacid sequences disclosed herein. It is well within the skill of a persontrained in the art to create these alternative DNA sequences encodingthe same, or essentially the same, toxins. These variant DNA sequencesare within the scope of the subject invention. As used herein, referenceto “essentially the same” sequence refers to sequences which have aminoacid substitutions, deletions, additions, or insertions which do notmaterially affect pesticidal activity. Fragments retaining pesticidalactivity are also included in this definition.

Alterations can be made at the amino or carboxy terminus of theinsecticidal proteins and variants of the invention that result inpolypeptides that retain biological activity. Fragments or biologicallyactive portions include polypeptide fragments comprising amino acidsequences sufficiently identical to the amino acid sequence set forth inSEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, and 40. A biologically active portion of a delta endotoxinprotein can be a polypeptide that is, for example, 10, 25, 50, 100, ormore amino acids in length. Such biologically active portions can beprepared by recombinant protein engineering techniques well known in theart and evaluated for insecticidal activity. Methods for measuringpesticidal activity are well known in the art. As used herein a fragmentencompasses at least 8 contiguous amino acids of SEQ ID NOs:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40. Theinvention encompasses other fragments, however, such as any fragment inthe protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1050, 1100, 1150, or 1200 amino acids, up to the full length of theinsecticidal proteins or variant proteins of SEQ ID NOs:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

Fragments with improved biological activity, pest spectrum, or theability to control resistant insect populations are also provided in thepresent invention. Modifications can be made to Cry proteins to producefragments with improved pore formation and thereby pesticidal activity.In the case of three-domain Cry proteins, domain 1 is comprised of sevenα-helices involved in pore formation in the midgut of susceptibleinsects. Modified DIG proteins with improved activity can be designed tohave N-terminal deletions in regions with putative secondary structurehomology to α-helix 1 and α-helix 2 of domain 1.

Proteases may be used to directly obtain active fragments of thesetoxins. A fragment of a claimed insecticidal toxin will comprise atleast about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100 or 1200contiguous amino acids, or up to the total number of amino acids presentin a full-length insecticidal toxin of the invention (for example, 625amino acids for SEQ ID NO:2, or 625 amino acids for SEQ ID NO:4).

Core toxin and Protoxin chimeras. A majority of Bacillus thuringiensisdelta-endotoxin crystal protein molecules are composed of two functionalsegments. The protease-resistant core toxin is the first segment andcorresponds to about the first half of the protein molecule. Theapproximately C-terminal half of the molecule is the second segment. Forpurposes of this application, this second segment will be referred toherein as the “protoxin segment.” The protoxin segment is believed toparticipate in toxin crystal formation (Arvidson et al., (1989)). Thefull 130 kDa toxin molecule is rapidly processed to the resistant coresegment by protease in the insect gut. The protoxin segment may thusconvey a partial insect specificity for the toxin by limiting theaccessibility of the core to the insect by reducing the proteaseprocessing of the toxin molecule (Haider et al., (1986)) or by reducingtoxin solubility (Aronson et al., (1991)).

Chimeric proteins advantageously joined within the toxin domains ofCry1Fa and Cry1Ab have been reported (U.S. Pat. No. 5,527,883). Othersuccess in the area has been reported in the literature. For example,the construction of hybrid delta-endotoxins is reported in the followingrelated art. Intl. Pat. Appl. Publ. No. WO 95/30753 discloses theconstruction of hybrid B. thuringiensis delta-endotoxins for productionin Pseudomonas fluorescens in which the non-toxic protoxin fragment ofCry1F has been replaced by the non-toxic protoxin fragment from theCry1Ac/Cry1Ab that is disclosed in U.S. Pat. No. 5,128,130. That patentalso discloses the construction of hybrid B. thuringiensisdelta-endotoxins for production in P. fluorescens in which a portion ofthe non-toxic protoxin segment of Cry1Ac is replaced with thecorresponding non-toxic protoxin fragment of Cry1Ab. U.S. Pat. No.5,055,294 discloses the construction of a specific hybriddelta-endotoxin between Cry1Ac (amino acid residues 1-466) and Cry1Ab(amino acid residues 466-1155) for production in P. fluorescens.Although the aforementioned patent discloses the construction of ahybrid toxin within the active toxin segment, no specifics are presentedin regard to the hybrid toxin's insecticidal activity. InternationalPatent Application Publication No. WO 95/30752 discloses theconstruction of hybrid B. thuringiensis delta-endotoxins for productionin P. fluorescens in which the non-toxic protoxin segment of Cry1C isreplaced by the non-toxic protoxin segment from Cry1Ab. Theafore-mentioned application further discloses that the activity againstSpodoptera exigua for the hybrid delta-endotoxin is improved over thatof the parent active toxin, Cry1C. International Patent ApplicationPublication No. WO 95/06730 discloses the construction of a hybrid B.thuringiensis delta-endotoxin consisting of domains 1 and 2 of Cry1Ecoupled to domain 3 and the non-toxic protoxin segment of Cry1C. Insectbioassays performed against Manduca sexta (sensitive to Cry1C andCry1E), Spodoptera exigua (sensitive to Cry1C), and Mamestra brassicae(sensitive to Cry1C) show that the hybrid Cry1E/Cry1C hybrid toxin isactive against M. sexta, S. exigua, and M. brassicae. The bioassayresults were expressed as EC₅₀ values (toxin concentration giving a 50%growth reduction) rather than LC₅₀ values (toxin concentration giving50% mortality). Although the delta-endotoxins used for bioassay wereproduced in B. thuringiensis, only artificially-generated activesegments of the delta-endotoxins were used, not the naturally-producedcrystals typically produced by B. thuringiensis that are present incommercial B. thuringiensis formulations. Bioassay results indicatedthat the LC₅₀ values for the hybrid Cry1E/Cry1C crystal against S.frugiperda were 1.5 to 1.7 fold lower (i.e. were more active) than fornative Cry1C. This art also discloses the construction of a hybrid B.thuringiensis delta-endotoxin between Cry1Ab (domains 1 and 2) and Cry1C(domain 3 and the non-toxic protoxin segment), although no data aregiven regarding the hybrid toxin's activity or usefulness.

Lee et al., (1995) report the construction of hybrid B. thuringiensisdelta-endotoxins between Cry1Ac and Cry1Aa within the active toxinsegment. Artificially generated active segments of the hybrid toxinswere used to examine protein interactions in susceptible insect brushborder membranes vesicles (BBMV). The bioactivity of the hybrid toxinswas not reported. Honee et al., (1991) report the construction of hybriddelta-endotoxins between Cry1C (domain 1) and Cry1Ab (domains 2 and 3)and the reciprocal hybrid between Cry1Ab (domain 1) and Cry1C (domains 2and 3). These hybrids failed to show any significant increase inactivity against susceptible insects. Furthermore, the Cry1C (domain1)/Cry1Ab (domains 2 and 3) hybrid toxin was found to be hypersensitiveto protease degradation. A report by Schnepf et al., (1990) disclosesthe construction of Cry1Ac hybrid toxin in which a small portion ofdomain 2 was replaced by the corresponding region of Cry1Aa, although nosignificant increase in activity against susceptible insect larvae wasobserved.

The chimeric toxins of the subject invention comprise a full coreN-terminal toxin portion of a B.t. toxin and, at some point past the endof the toxin portion, the protein has a transition to a heterologousprotoxin sequence. The transition to the heterologous protoxin segmentcan occur at approximately the native toxin/protoxin junction or aportion of the native protoxin (extending past the toxin portion) can beretained with the transition to the heterologous protoxin occurringdownstream. For example, a chimeric toxin of the subject invention mayhave the full toxin portion of a modified Cry1Ca toxin such as aminoacids 1-628 of DIG-473 or DIG-465 and a heterologous protoxin segment(amino acids 629 to the C-terminus). In a preferred embodiment, theheterologous protoxin segment portion is taken from Cry1Ab.

A person skilled in this art will appreciate that B.t. toxins, evenwithin a certain class, will vary to some extent in length and in theprecise location of the transition from the core toxin portion toprotoxin portion. The transition from core toxin portion to protoxinportion will typically occur at between about 50% to about 60% of thefull length toxin. The chimeric toxin of the subject invention includesthe full expanse of this core N-terminal toxin portion, such is the full628 amino acid length of RDIG544.12 insecticidal toxin protein. SEQ IDNO:15 discloses the 1887 nucleotide sequence of the DIG-465-encodingDNA, of which the 5′-terminal 1887 nucleotides comprise the codingregion for the core toxin segment of Cry1Ca with a mutation L57A(leucine at amino acid position 57 substituted for alanine), oneembodiment of the subject invention. SEQ ID NO:16 discloses the 628amino acid sequence of the full-length DIG-465 polypeptide, of which theN-terminal core portion of Cry1Ca with the above mentioned amino acidsubstitutions. SEQ ID NO:23 discloses the 1887 nucleotide sequence ofDIG-473-encoding DNA, which comprises the coding region for the coretoxin segment of Cry1Ca with a mutation F596M (phenylalanine at aminoacid position 596, substituted for methionine), another subject of theinvention. SEQ ID NO:24 discloses the 628 amino acid sequence of thefull-length DIG-473 polypeptide, which comprises the portion of Cry1Cawith the above mentioned amino acid substitutions.

With regard to the protoxin portion, the full expanse of the nativeCry1Ab protoxin portion extends from the end of the toxin portion of theCry1Ab full length protein to the C-terminus of the molecule. Attentionis drawn to the last about 100 to 150 amino acids of this protoxin,which are most critical to include in the chimeric toxin of the subjectinvention.

Because Cry proteins have selective insecticidal activity, most areactive on a limited range of target pests. There is, therefore, a needto further improve the biological activity attributes of Cry proteins.Cry proteins with unique binding characteristics and modes of action areuseful in strategies to expand the range of insect pests controlled orcounter the development of B.t. resistance.

Domain III modifications. As described herein, the B.t. insecticidaltoxins of the subject invention are 3-domain type toxins, comprisingDomain I, Domain II, and Domain III. Domain III binds certain classes ofreceptor proteins and perhaps participates in insertion of an oligomerictoxin pre-pore. Specific hybrid toxins that comprised domain IIIsubstitutions were shown to have superior toxicity against Spodopteraexigua (de Maagd et al., 1996) and guidance exists on the design of theCry toxin domain swaps (Knight et al., 2004).

Domain I modifications. Numerous studies using biochemical and molecularapproaches have provided information on the determinants of Cry proteinbinding and insertion into insect midgut membranes (reviewed in Piggotand Ellar, 2007). Domain I from Cry1A and Cry3A proteins has beenstudied for the ability to insert and form pores in membranes. α-helices4 and 5 of domain I play key roles in membrane insertion and poreformation (Walters et al., 1993, Gazit et al., 1998; Nunez-Valdez etal., 2001), with the other helices proposed to contact the membranesurface like the ribs of an umbrella (Gazit et al., 1998).

Alpha-helix 3 appears in some instances to be required for oligomericpre-pore formation and toxicity. Some α-helix 3 mutants are able to bindreceptors but do not form oligomers and are non-toxic to Manduca sexta(reviewed in Jimenez-Juarez et al., 2008). However, proteolyticallyactivated forms of Cry3Aa1 lack α-helices 1, 2 and 3 (Carroll et al.,1997).

Alpha-helix 1 is removed following receptor binding. Gomez et al.,(2002) found that Cry1Ab oligomers formed upon BBMV receptor bindinglacked the α-helix 1 portion of domain I. Also, Soberon et al., (2007)have shown that N-terminal deletion mutants of Cry1Ab and Cry1Ac whichlack approximately 60 amino acids encompassing α-helix 1 on the threedimensional Cry structure are capable of assembling monomers ofmolecular weight about 60 kDa into pre-pores in the absence of cadherinbinding. These N-terminal deletion mutants were reported to be active onCry-resistant insect larvae. Furthermore, Diaz-Mendoza et al., (2007)described Cry1Ab fragments of 43 kDa and 46 kDa that retained activityon Mediterranean corn borer (Sesamia nonagrioides). These fragments weredemonstrated to include amino acid residues 116 to 423; however theprecise amino acid sequences were not elucidated and the mechanism ofactivity of these proteolytic fragments is unknown. The results of Gomezet al., (2002), Soberon et al., 2007 and Diaz-Mendoza et al., (2007)contrast with those of Hofte et al., (1986), who reported that deletionof 36 amino acids from the N-terminus of Cry1Ab resulted in loss ofinsecticidal activity.

Anti-toxin antibodies. Equivalent toxins and/or genes encoding theseequivalent toxins can be derived from Bacillus thuringiensis isolatesand/or DNA libraries using the teachings provided herein. There are anumber of methods for obtaining the pesticidal toxins of the instantinvention. For example, antibodies immunoreactive to the pesticidaltoxins disclosed and claimed herein can be used to identify and isolateother toxins from a mixture of proteins. Specifically, antibodies may beraised to the portions of the toxins which are most constant and mostdistinct from other B.t. toxins. These antibodies can then be used tospecifically identify equivalent toxins with the characteristic activityby, for example, immunoprecipitation, enzyme linked immunosorbent assay(ELISA), or immunoblotting (western blotting). Antibodies to the toxinsdisclosed herein, or to equivalent toxins, or fragments of these toxins,can readily be prepared using standard procedures in this art. The geneswhich encode these toxins can then be obtained from the microorganismsthat produce the toxins.

Once the B.t. insecticidal toxin has been isolated, antibodies specificfor the toxin may be raised by conventional methods that are well knownin the art. Repeated injections into a host of choice over a period ofweeks or months will elicit an immune response and result in significantanti-B.t. toxin serum titers. Preferred hosts are mammalian species andmore highly preferred species are rabbits, goats, sheep, and mice. Blooddrawn from such immunized animals may be processed by establishedmethods to obtain antiserum (polyclonal antibodies) reactive with theB.t. insecticidal toxin. The antiserum may then be affinity purified byadsorption to the toxin according to techniques known in the art.Affinity purified antiserum may be further purified by isolating theimmunoglobulin fraction within the antiserum using procedures known inthe art. The resulting material will be a heterogeneous population ofimmunoglobulins reactive with the B.t. insecticidal toxin.

Anti-B.t. toxin antibodies may also be generated by preparing asemi-synthetic immunogen consisting of a synthetic peptide fragment ofthe B.t. insecticidal toxin conjugated to an immunogenic carrier.Numerous schemes and instruments useful for making peptide fragments arewell known in the art. Many suitable immunogenic carriers such as bovineserum albumin (BSA) or keyhole limpet hemocyanin are also well known inthe art, as are techniques for coupling the immunogen and carrierproteins. Once the semi-synthetic immunogen has been constructed, theprocedure for making antibodies specific for the B.t. insecticidal toxinfragment is identical to those used for making antibodies reactive withnatural B.t. toxin.

Anti-B.t. toxin monoclonal antibodies (MAbs) are readily prepared usingpurified B.t. insecticidal toxin. Methods for producing MAbs have beenpracticed for over 15 years and are well known to those of ordinaryskill in the art. Repeated intraperitoneal or subcutaneous injections ofpurified B.t. insecticidal toxin in adjuvant will elicit an immuneresponse in most animals. Hyperimmunized B-lymphocytes are removed fromthe animal and fused with a suitable fusion partner cell line capable ofbeing cultured indefinitely. Preferred animals whose B-lymphocytes maybe hyperimmunized and used in the production of MAbs are mammals. Morepreferred animals are rats and mice and most preferred is the BALB/cmouse strain.

Numerous mammalian cell lines are suitable fusion partners for theproduction of hybridomas. Many such lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.) and commercialsuppliers. Preferred fusion partner cell lines are derived from mousemyelomas and the HL-1@ Friendly myeloma-653 cell line (Ventrex,Portland, Me.) is most preferred. Once fused, the resulting hybridomasare cultured in a selective growth medium for one to two weeks. Two wellknown selection systems are available for eliminating unfused myelomacells, or fusions between myeloma cells, from the mixed hybridomaculture. The choice of selection system depends on the strain of mouseimmunized and myeloma fusion partner used. The AAT selection system,described by Taggart and Samloff (1983), may be used; however, the HAT(hypoxanthine, aminopterin, thymidine) selection system, described byLittlefield (1964), is preferred because of its compatibility with thepreferred mouse strain and fusion partner mentioned above. Spent growthmedium is then screened for immunospecific MAb secretion. Enzyme linkedimmunosorbent assay (ELISA) procedures are best suited for this purpose;though, radioimmunoassays adapted for large volume screening are alsoacceptable. Multiple screens designed to consecutively pare down theconsiderable number of irrelevant or less desired cultures may beperformed. Cultures that secrete MAbs reactive with the B.t.insecticidal toxin may be screened for cross-reactivity with known B.t.insecticidal toxins. MAbs that preferentially bind to the preferred B.t.insecticidal toxin may be isotyped using commercially available assays.Preferred MAbs are of the IgG class, and more highly preferred MAbs areof the IgG1 and IgG2a subisotypes.

Hybridoma cultures that secrete the preferred MAbs may be sub-clonedseveral times to establish monoclonality and stability. Well knownmethods for sub-cloning eukaryotic, non-adherent cell cultures includelimiting dilution, soft agarose and fluorescence activated cell sortingtechniques. After each subcloning, the resultant cultures preferably arere-assayed for antibody secretion and isotype to ensure that a stablepreferred MAb-secreting culture has been established.

The anti-B.t. toxin antibodies are useful in various methods ofdetecting the claimed B.t. insecticidal toxin of the instant invention,and variants or fragments thereof. It is well known that antibodieslabeled with a reporting group can be used to identify the presence ofantigens in a variety of milieus. Antibodies labeled with radioisotopeshave been used for decades in radioimmunoassays to identify, with greatprecision and sensitivity, the presence of antigens in a variety ofbiological fluids. More recently, enzyme labeled antibodies have beenused as a substitute for radiolabeled antibodies in the ELISA assay.Further, antibodies immunoreactive to the B.t. insecticidal toxin of thepresent invention can be bound to an immobilizing substance such as apolystyrene well or particle and used in immunoassays to determinewhether the B.t. toxin is present in a test sample.

In one preferred embodiment, insecticidal proteins or a variant isdelivered orally through a transgenic plant comprising a nucleic acidsequence that expresses a toxin of the present invention. The presentinvention provides a method of producing an insect-resistant transgenicplant, comprising introducing a nucleic acid molecule of the inventioninto the plant wherein the toxin is expressible in the transgenic plantin an effective amount to control an insect. In a non-limiting example,a basic cloning strategy may be to subclone full length or modified Crycoding sequences (CDS) into a plant expression plasmid at NcoI and SacIrestriction sites. The resulting plant expression cassettes containingthe appropriate Cry coding region under the control of plant expressionelements, (e.g., plant expressible promoters, 3′ terminal transcriptiontermination and polyadenylate addition determinants, and the like) aresubcloned into a binary vector plasmid, utilizing, for example, Gateway®technology or standard restriction enzyme fragment cloning procedures.LR Clonase™ (Invitrogen) for example, may be used to recombine the fulllength and modified gene plant expression cassettes into a binary planttransformation plasmid if the Gateway® technology is utilized. It isconvenient to employ a binary plant transformation vector that harbors abacterial gene that confers resistance to the antibiotic spectinomycinwhen the plasmid is present in E. coli and Agrobacterium cells. It isalso convenient to employ a binary vector plasmid that contains aplant-expressible selectable marker gene that is functional in thedesired host plants. Examples of plant-expressible selectable markergenes include but are not limited to aminoglycoside phosphotransferasegene of transposon Tn5 (Aph II) which encodes resistance to theantibiotics kanamycin, neomycin and G418, as well as those genes whichcode for tolerance to glyphosate; hygromycin; methotrexate;phosphinothricin (bialaphos), imidazolinones, sulfonylureas andtriazolopyrimidine herbicides, such as chlorosulfuron, bromoxynil,dalapon and the like.

Alternatively, the plasmid structure of the binary plant transformationvector containing the DIG-465, DIG-473, DIG-468, DIG-483, DIG-462,DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485,DIG-487, IRDIG544.8, IRDIG544.9, IRDIG544.11, or IRDIG544.12 gene insertis performed by restriction digest fingerprint mapping of plasmid DNAprepared from candidate Agrobacterium isolates by standard molecularbiology methods well known to those skilled in the art of Agrobacteriummanipulation.

Those skilled in the art of obtaining transformed plants viaAgrobacterium-mediated transformation methods will understand that otherAgrobacterium strains besides Z707S may be used, and the choice ofstrain may depend upon the identity of the host plant species to betransformed.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted. All patents, patent applications,provisional applications, and publications referred to or cited hereinare incorporated by reference in their entirety to the extent they arenot inconsistent with the explicit teachings of this specification.Unless specifically indicated or implied, the terms “a”, “an”, and “the”signify “at least one” as used herein.

Example 1 Design of a Plant-Optimized Version of the Coding Sequence forB.t. Insecticidal Proteins

A DNA sequence having a plant codon bias was designed and synthesized toproduce the insecticidal proteins in transgenic monocot and dicotplants. A codon usage table for maize (Zea mays L.) was calculated from706 protein coding sequences (CDS) obtained from sequences deposited inGenBank. Codon usage tables for tobacco (Nicotiana tabacum, 1268 CDS),canola (Brassica napus, 530 CDS), cotton (Gossypium hirsutum, 197 CDS),and soybean (Glycine max; ca. 1000 CDS) were downloaded from data at thewebsite http://www.kazusa.or.jp/codon/. A biased codon set thatcomprises highly used codons common to both maize and dicot datasets, inappropriate weighted average relative amounts, was calculated afteromitting any redundant codon used less than about 10% of total codonuses for that amino acid in either plant type. To derive a plantoptimized sequence encoding the insecticidal protein, codonsubstitutions to the insecticidal protein DNA sequences were made suchthat the resulting DNA sequence had the overall codon composition of theplant-optimized codon bias table. Further refinements of the sequencewere made to eliminate undesirable restriction enzyme recognition sites,potential plant intron splice sites, long runs of A/T or C/G residues,and other motifs that might interfere with RNA stability, transcription,or translation of the coding region in plant cells. Other changes weremade to introduce desired restriction enzyme recognition sites, and toeliminate long internal Open Reading Frames (frames other than +1).These changes were all made within the constraints of retaining theplant-biased codon composition. To complete the design, a sequenceencoding translational Stop codons in all 6 open reading frames wasadded to the 3′ end of the coding regions, and appropriate restrictionrecognition sites were added to the 5′ and 3′ ends of the sequences.Synthesis of the designed sequence was performed by a commercial vendor(DNA2.0, Menlo Park, Calif.). Additional guidance regarding theproduction of synthetic genes can be found in, for example, WO 97/13402and U.S. Pat. No. 5,380,831.

Plant-optimized DNA sequences encoding DIG proteins of the subjectinvention (SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, and 40) are disclosed as SEQ ID NOs:1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39. DNAmolecules comprising sequences disclosed in SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and 39 weresynthetically assembled by a commercial entity (DNA2.0).

Example 2 Construction of Expression Plasmids Encoding InsecticidalToxins and Expression in Bacterial Hosts

Standard cloning methods were used in the construction of Pseudomonasfluorescens (Pf) expression plasmids engineered to produce DIG-465 (SEQID NO: 16), DIG-473 (SEQ ID NO:24), DIG-468 (SEQ ID NO:2), DIG-483 (SEQID NO:4), DIG-462 (SEQ ID NO:10), DIG-463 (SEQ ID NO:12), DIG-464 (SEQID NO:14), DIG-466 (SEQ ID NO:18), DIG-467 (SEQ ID NO:20), DIG-469 (SEQID NO:22), DIG-474 (SEQ ID NO:26), DIG-482 (SEQ ID NO:28), DIG-485 (SEQID NO:6), and DIG-487 (SEQ ID NO:8) proteins encoded by plant-optimizedcoding regions. Restriction endonucleases were obtained from New EnglandBioLabs (NEB; Ipswich, Mass.) and T4 DNA Ligase (Invitrogen Corporation,Carlsbad, Calif.) was used for DNA ligation.

The basic cloning strategy entailed subcloning the DIG-465, DIG-473,DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469,DIG-474, DIG-482, DIG-485, or DIG-487 toxin coding sequence (CDS) intopDOW 1169 at restriction sites such as SpeI and XhoI, whereby it wasplaced under the expression control of the Ptac promoter and therrnBT1T2 terminator from plasmid pKK223-3 (PL Pharmacia, Milwaukee,Wis.). pDOW1169 is a low copy plasmid with the RSF1010 origin ofreplication, a pyrF gene, and a ribosome binding site preceding therestriction enzyme recognition sites into which DNA fragments containingprotein coding regions may be introduced, (US Patent ApplicationUS20080193974). The expression plasmid was transformed byelectroporation into DC454 (a near wild-type P. fluorescens strainhaving mutations ΔpyrF and lsc: lacIQI), or its derivatives, recoveredin SOC-Soy hydrolysate medium, and plated on selective medium (M9glucose agar lacking uracil, Sambrook et al., supra). Details of themicrobiological manipulations are available in Squires, C. H. et al.,(2004), US Patent Application 20060008877, US Patent Application20080193974, and US Patent Application 20080058262, incorporated hereinby reference. Strains were validated by restriction digestion ofminiprep plasmid DNA.

Growth and Expression Analysis in Shake Flasks. Production of DIG-465,DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467,DIG-469, DIG-474, DIG-482, DIG-485, or DIG-487 toxin forcharacterization and insect bioassay was accomplished byshake-flask-grown P. fluorescens strains harboring expressionconstructs. Seed cultures grown in M9 medium supplemented with 1%glucose and trace elements were used to inoculate 50 mL of definedminimal medium with 5% glycerol (Teknova Cat. #3D7426, Hollister,Calif.). Expression of the insecticidal protein toxin gene via the Ptacpromoter was induced by addition ofisopropyl-β-D-1-thiogalactopyranoside (IPTG) after an initial incubationof 24 hours at 30° C. with shaking. Cultures were sampled at the time ofinduction and at various times post-induction. Cell density was measuredby optical density at 600 nm (OD₆₀₀). Other culture media suitable forgrowth of Pseudomonas fluorescens may also be utilized, for example, asdescribed in Huang et al., 2007 and US Patent Application 20060008877.

Cell Fractionation and SDS-PAGE Analysis of Shake Flask Samples. At eachsampling time, 0.5 mL aliquots were centrifuged at 14000×g for fiveminutes. The cell pellets were frozen at −80° C. Soluble and insolublefractions from frozen shake flask cell pellet samples were generatedusing BugBuster Master Mix (EMDMillipore® Darmstadt, Germany). Each cellpellet was resuspended in 0.5 mL BugBuster Master Mix™ solution andincubated with shaking at room temperature for 30 minutes. The sampleswere lysed using a beadbeater with 0.1 mm glass beads for 3 minutes. Thelysate was centrifuged at 14,000 rpm for 5 minutes and the supernatantwas recovered as the soluble fraction. The pellet (insoluble fraction)was then resuspended in an equal volume of extraction buffer (8 M urea,0.5 M NaCl, 25 mM NaPO4, pH 10.4).

Samples were mixed 1:1 with 2×NuPAGE Tris Glycine SDS Sample Buffer(Invitrogen, Carlsbad, Calif.) containing dithiothreitol (DTT) andboiled for 5 minutes prior to loading onto Novex 4-20% Tris Glycine SDSpolyacrylamide gel (Invitrogen, Carlsbad, Calif.). Electrophoresis wasperformed in the recommended Tris-Glycine buffer. Gels were stained withBio-Safe Coomassie Stain according to the manufacturer's (Bio-Rad Inc.,Hercules, Calif.) protocol and imaged using the GE Typhooon SeriesImaging system (Pittsburgh, Pa.).

Inclusion body preparation. Cry protein inclusion body (IB) preparationswere performed on cells from P. fluorescens fermentations that producedinsoluble B.t. insecticidal protein, as demonstrated by SDS-PAGE andMALDI-MS (Matrix Assisted Laser Desorption/Ionization MassSpectrometry). P. fluorescens fermentation pellets were thawed in a 37°C. water bath. The cells were resuspended to 25% w/v in lysis buffer (50mM Tris, pH 7.5, 200 mM NaCl, 20 mM EDTA disodium salt(Ethylenediaminetetraacetic acid), 1% Triton X-100, and 5 mMDithiothreitol (DTT); 5 mL/L of bacterial protease inhibitor cocktail(P8465 Sigma-Aldrich, St. Louis, Mo.) were added just prior to use). Thecells were suspended using a hand-held homogenizer at the lowest setting(Tissue Tearor, BioSpec Products, Inc Bartlesville, Okla.). Lysozyme (25mg of Sigma-Aldrich L7651, from chicken egg white) was added to the cellsuspension by mixing with a metal spatula, and the suspension wasincubated at room temperature for one hour. The suspension was cooled onice for 15 minutes, then sonicated using a Branson Sonifier 250 (two1-minute sessions, at 50% duty cycle, 30% output). Cell lysis waschecked by microscopy. An additional 25 mg of lysozyme was added ifnecessary, and the incubation and sonication were repeated. When celllysis was confirmed via microscopy, the lysate was centrifuged at11,500×g for 25 minutes (4° C.) to form the IB pellet, and thesupernatant was discarded. The IB pellet was suspended with 100 mL lysisbuffer, homogenized with the hand-held mixer and centrifuged as above.The IB pellet was repeatedly washed by suspension (in 50 mL lysisbuffer), homogenization, sonication, and centrifugation until thesupernatant became colorless and the IB pellet became firm and off-whitein color. For the final wash, the IB pellet was suspended insterile-filtered (0.22 μm) distilled water containing 2 mM EDTA, andcentrifuged. The final pellet was suspended in sterile-filtereddistilled water containing 2 mM EDTA, and stored in 1 mL aliquots at−80° C.

SDS-PAGE analysis and quantification of protein in IB preparations wasdone by thawing a 1 mL aliquot of IB pellet and diluting 1:20 withsterile-filtered distilled water. The diluted sample was then boiledwith 4× reducing sample buffer [250 mM Tris, pH 6.8, 40% glycerol (v/v),0.4% Bromophenol Blue (w/v), 8% SDS (w/v) and 8% β-Mercapto-ethanol(v/v)] and loaded onto a Novex® 4-20% Tris-Glycine, 12+2 well gel(Invitrogen) run with 1× Tris/Glycine/SDS buffer (BioRad). The gel wasrun for 60 min at 200 volts then stained with Coomassie Blue (50%G-250/50% R-250 in 45% methanol, 10% acetic acid), and destained with 7%acetic acid, 5% methanol in distilled water. Quantification of targetbands was done by comparing densitometric values for the bands againstBovine Serum Albumin (BSA) samples run on the same gel to generate astandard curve.

Solubilization of Inclusion Bodies. Six mL of inclusion body suspensionfrom Pf clone containing DIG-465, DIG-473, DIG-468, DIG-483, DIG-462,DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485,or DIG-487 protein were centrifuged on the highest setting of anEppendorf model 5415C microfuge (approximately 14,000×g) to pellet theinclusions. The storage buffer supernatant was removed and replaced with25 mL of 100 mM sodium carbonate buffer, pH 11, in a 50 mL conical tube.Inclusions were resuspended using a pipette and vortexed to mixthoroughly. The tube was placed on a gently rocking platform at 4° C.overnight to extract the target protein. The extract was centrifuged at30,000×g for 30 min at 4° C., and the resulting supernatant wasconcentrated 5-fold using an Amicon Ultra-15 regenerated cellulosecentrifugal filter device (30,000 Molecular Weight Cutoff, Millipore).The sample buffer was then changed to 10 mM CAPS[3-(cyclohexamino)1-propanesulfonic acid] pH 10, using disposable PD-10columns (GE Healthcare, Piscataway, N.J.).

Gel electrophoresis. The concentrated extract was prepared forelectrophoresis by diluting 1:50 in NuPAGE® LDS sample buffer(Invitrogen) containing 5 mM dithiothreitol as a reducing agent andheated at 95° C. for 4 minutes. The sample was loaded in duplicate lanesof a 4-12% NuPAGE® gel alongside five BSA standards ranging from 0.2 to2 μg/lane (for standard curve generation). Voltage was applied at 200Vusing MOPS SDS running buffer (Invitrogen) until the tracking dyereached the bottom of the gel. The gel was stained with 0.2% CoomassieBlue G-250 in 45% methanol, 10% acetic acid, and destained, firstbriefly with 45% methanol, 10% acetic acid, and then at length with 7%acetic acid, 5% methanol until the background cleared. Followingdestaining, the gel was scanned with a Biorad Fluor-S MultiImager. Theinstrument's Quantity One v.4.5.2 Software was used to obtainbackground-subtracted volumes of the stained protein bands and togenerate the BSA standard curve that was used to calculate theconcentration of DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463,DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485, orDIG-487 protein in the stock solution.

The level of expression of DIG-465, DIG-473, DIG-468, DIG-483, DIG-463,DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485, andDIG-487 was compared to the level of expression of truncated Cry1Ca(DIG-462) when expressed in Pseudomonas fluorescens bacterial cells.Truncated Cry1Ca (DIG-462) expressed at approximately 1 g/1, whereasDIG-473 expressed at approximately 0.5 g/1. DIG-465 expressed atapproximately 5-fold higher than truncated Cry1Ca, at 4.9 g/1. These invitro results show that the L57A mutation results in greater expressionof truncated Cry1Ca protein.

Example 3 Insecticidal Activity of DIG Proteins Produced in Pseudomonasfluorescens

B.t. insecticidal toxins DIG-462, DIG-463, DIG-464, DIG-465, DIG-466,DIG-467, DIG-468, DIG-469, DIG-470, DIG-473, and DIG-474 weredemonstrated to be active on Lepidopteran species including diamondbackmoth (DBM; Plutella xylostella (Linnaeus)) and fall armyworm (FAW,Spodoptera frugiperda (Smith)).

Sample preparation and bioassays. Inclusion body preparations in 10 mMCAPS pH10 were diluted appropriately in 10 mM CAPS, pH 10, and allbioassays contained a control treatment consisting of this buffer, whichserved as a background check for mortality or growth inhibition.

Protein concentrations in bioassay buffer were estimated by gelelectrophoresis using BSA to create a standard curve for geldensitometry, which was measured using a BioRad imaging system (Fluor-SMultiImager with Quantity One software version 4.5.2). Proteins in thegel matrix were stained with Coomassie Blue-based stain and destainedbefore reading.

Purified proteins were tested for insecticidal activity in bioassaysconducted with neonate Lepidopteran larvae on artificial insect diet.Larvae of DBM and FAW were hatched from eggs obtained from a colonymaintained by a commercial insectary (Benzon Research Inc., Carlisle,Pa.). Larvae of rFAW were hatched from eggs harvested from a proprietarycolony (Dow AgroSciences LLC, Indianapolis, Ind.).

These bioassays were conducted in 128-well plastic trays specificallydesigned for insect bioassays (C-D International, Pitman, N.J.). Eachwell contained 1.0 mL of multi-species Lepidoptera diet (SouthlandProducts, Lake Village, Ark.). A 40 μL aliquot of protein sample wasdelivered by pipette onto the 1.5 cm² diet surface of each well (26.7μL/cm²). Cry protein concentrations were calculated as the amount (ng)of DIG protein per square centimeter (cm²) of surface area in the well.The treated trays were held in a fume hood until the liquid on the dietsurface had evaporated or was absorbed into the diet.

Within a few hours of eclosion, individual larvae were picked up with amoistened camel hair brush and deposited on the treated diet, one larvaper well. The infested wells were then sealed with adhesive sheets ofclear plastic, vented to allow gas exchange (C-D International, Pitman,N.J.). Bioassay trays were held under controlled environmentalconditions (28° C., ˜60% Relative Humidity, 16:8 [Light:Dark]) for 5days, after which the total number of insects exposed to each proteinsample, the number of dead insects, and the weight of surviving insectswere recorded. Percent mortality and percent growth inhibition werecalculated for each treatment. Growth inhibition (GI) was calculated asfollows:

GI=[1−(TWIT/TNIT)/(TWIBC/TNIBC)]

where TWIT is the Total Weight of Insects in the Treatment,

TNIT is the Total Number of Insects in the Treatment

TWIBC is the Total Weight of Insects in the Background Check (Buffercontrol), and

TNIBC is the Total Number of Insects in the Background Check (Buffercontrol).

In the DBM bioassay, 10 and 300 ng/cm² of DIG-462, DIG-463, DIG-464,DIG-465, DIG-466, DIG-467, DIG-468, DIG-469, DIG-470, DIG-471, DIG-472,DIG-473, and DIG-474 were tested against the insect sp. The FAW weretested with inclusion body preparation of DIG-462, DIG-465, DIG-473 at1× and 5× dilution rate. Percent mortality and growth inhibition resultswere compared.

Mortality was 100% at 300 ng/cm² for DIG-462, DIG-463, DIG-464, DIG-465,DIG-466, DIG-468, DIG-469, DIG-473, and DIG-474 treatments (Table 2 andTable 3). Growth inhibition was 70-90% inhibition of growth at 10 ng/cm²and 100% inhibition at 300 ng/cm² for DIG-465 and DIG-473 treatments(Table 2).

TABLE 2 Results of bioassay tests of DIG-462, DIG-465 and DIG-473proteins on DBM, measuring both mortality and growth inhibition ProteinMortality Growth Inhibition DIG-462 +++ ++++ DIG-465 ++ ++++ DIG-473 +++++++ For Mortality ++ = 0-20% at 10 ng/cm² and 100% at 300 ng/cm², +++ =30-60% at 10 ng/cm² and 100% at 300 ng/cm². For Growth Inhibition ++++ =70-90% inhibition of growth at 10 ng/cm² and 100% inhibition at 300ng/cm².

TABLE 3 Bioassay results of protein mutants tested against DBM at 10ng/cm² and 300 ng/cm² concentrations. % Mortality % Mortality Protein at10 ng/cm² at 300 ng/cm² DIG-462 38 100 DIG-463 63 100 DIG-464 38 100DIG-465 0 100 DIG-466 38 100 DIG-467 38 88 DIG-468 13 100 DIG-469 0 100DIG-470 0 50 DIG-471 0 0 DIG-472 0 0 DIG-473 38 100 DIG-474 0 100 BSA 00

Growth inhibition of Cry1Ca core toxin (DIG-462), DIG-465, and DIG-473protein to FAW larvae was determined to be >40% for all treatments(Table 4). Proteins were tested at full strength and diluted 5-fold withbuffer (10 mM CAPS, pH 10).

TABLE 4 Percent growth inhibition of DIG- 462, DIG-465, and DIG-473 toFAW Protein Dilution % Growth Inhibition DIG-462 1X 43 DIG-462 5X 47DIG-465 1X 81 DIG-465 5X 58 DIG-473 1X 56 DIG-473 5X 48 Buffer 1X 0

DBM activity and the susceptibility of the purified protein to bedigested by chymotrypsin were assessed. An unexpected and surprisingfinding was that DIG-473 was resistant to chymotrypsin cleavage while atthe same time having the same potency against DBM as DIG-462. This is incontrast to the Cry1Ca core (DIG-462) and DIG-465 proteins, which weresusceptible to chymotrypsin cleavage in vitro (Table 5).

TABLE 5 Proteins with activity against DBM (DIG-462 is the standard) andprotein resistance to cleavage by Chymotrypsin. Activity Resistant toDIG # Mutation Type on DBM Chymotrypsin 462 truncated ++++ No 463 G54A++++ No 464 L57M ++++ No 465 L57A ++++ No 466 V68F ++++ No 467 V68I ++++No 468 ΔGPS +++ No 469 W73A +++ Partial 473 F596M ++++ Yes 474 F596A++++ No 482 G54A/W73M +++ Yes 483 G54A/ΔGPS +++ Yes 485 L57A/ΔGPS +++ No487 L57M/ΔGPS +++ No

Example 4 European Corn Borer (ECB), Southwestern Corn Borer (SWCB) andSouthern Armyworm (SAW) Bioassays

Bioassays were conducted in 32-well test trays. Approximately 5 mL of a2% water-agar solution was applied to each well and the agar was allowedto solidify completely.

Plants were approximately 3 weeks old and tested at T₁ generation. Threereplicates of T₁ leaf material were completed. One leaf was cut (1″×0.5″rectangular) and placed in a single well of the tray. Each well wasinfested with 10 individual insect larvae (usually less than 24 hoursold) of the ECB, Cry1Fa rECB or SWCB. For SAW, 5 individual insectlarvae were infested per well. Seed based plants originated from B104inbred lines and Yellow Fluorescent Protein (YFP) transformed plantsserved as negative controls.

The infested wells were then sealed with adhesive sheets of clearplastic, vented to allow gas exchange (C-D International, Pitman, N.J.).Trays were placed in a conviron incubator and maintained at 28° C. (16:8h light:dark, 60% RH) for 3 days, after which the total amount of damageto each leaf piece (0, 5, 10, 15, 25, 50, 75% damage, etc., up to 100%)was recorded.

There was reduced feeding damage caused by ECB and Cry1Fa resistant ECB(rECB) when the insect larvae were exposed to plants containingtruncated Cry1Ca modified protein. When tested in a diet bioassay, wherepurified full length Cry1Ca is placed on top of an artificial insectdiet and individual insects are allowed to feed on the diet containingthe toxin, modified Cry1Ca is found to be inactive against ECB and rECB.However, when expressed in maize, at concentrations of >120 ng/cm², theexpression of Cry1Ca in the plant provides unexpected protection againstfeeding damage caused by ECB and especially rECB.

TABLE 6 Bioassay results of IRDIG544.12 T₁ maize when fed to EuropeanCorn Borer (ECB) and Cry1Fa-resistant ECB (rECB) ECB Avg. rECB Avg.Plant Name Description Damage Damage Toxinng/cm² YFP negative controlcontrol 100 88.3 0 YFP negative control control 100 93.3 0 YFP negativecontrol control 97.7 98.3 0 YFP negative control control 99.3 98 0 YFPnegative control control 100 92.7 0 114269[1]-021.001AJ.025 IRDIG544.1296 85 41 w/TraP12 114269[1]-021.001AJ.018 IRDIG544.12 94.3 90 42w/TraP12 114269[1]-021.001AJ.017 IRDIG544.12 99.3 95 34 w/TraP12114269[1]-021.001AJ.023 IRDIG544.12 99.3 50 36 w/TraP12114269[1]-021.001AJ.016 IRDIG544.12 99.3 80 33 w/TraP12114260[1]-021.AJ001.023 IRDIG544.12 66.7 11.7 210114260[1]-021.AJ001.029 IRDIG544.12 69.7 13.3 230114260[1]-021.AJ001.021 IRDIG544.12 66.7 16.7 210114260[1]-021.AJ001.016 IRDIG544.12 50 18.3 230 114260[1]-021.AJ001.019IRDIG544.12 50 18.3 210 114259[1]-009.AJ001.018 IRDIG544.12 95 85 100114259[1]-009.AJ001.022 IRDIG544.12 86 61.7 130 114259[1]-009.AJ001.021IRDIG544.12 93.3 75 140 114259[1]-009.AJ001.026 IRDIG544.12 80 95 67114259[1]-009.AJ001.027 IRDIG544.12 100 61.7 92 114260[1]-010.001AJ.054IRDIG544.12 68.3 25 180 114260[1]-010.001AJ.048 IRDIG544.12 71.7 18.3180 114260[1]-010.001AJ.047 IRDIG544.12 66.7 50 140114260[1]-010.001AJ.052 IRDIG544.12 71.7 20 220 114260[1]-010.001AJ.046IRDIG544.12 86.7 16.7 200 114269[1]-029.001AJ.027 IRDIG544.12 91 66.7 39w/TraP12 114269[1]-029.001AJ.028 IRDIG544.12 88.3 66.7 30 w/TraP12114269[1]-029.001AJ.023 IRDIG544.12 96.7 88.3 41 w/TraP12114269[1]-029.001AJ.026 IRDIG544.12 96 76.7 37 w/TraP12114269[1]-029.001AJ.019 IRDIG544.12 96.7 95 41 w/TraP12114267[1]-009.001AJ.044 IRDIG544.12 95 70 36 w/TraP12114267[1]-009.001AJ.034 IRDIG544.12 88.3 56.7 44 w/TraP12114267[1]-009.001AJ.032 IRDIG544.12 96.7 86.7 41 w/TraP12114267[1]-009.001AJ.037 IRDIG544.12 96.7 88.3 43 w/TraP12114267[1]-009.001AJ.030 IRDIG544.12 91.7 80 43 w/TraP12114259[1]-006.001AJ.015 IRDIG544.12 91.7 90 38 114259[1]-006.001AJ.014IRDIG544.12 98.3 85 41 114259[1]-006.001AJ.005 IRDIG544.12 95 80 40114259[1]-006.001AJ.010 IRDIG544.12 81.7 86 39 114259[1]-006.001AJ.013IRDIG544.12 85 88.3 47 114270[1]-027.AJ001.029 IRDIG544.12 98.7 96.7 170w/TraP12 114270[1]-027.AJ001.030 IRDIG544.12 91.7 99.3 150 w/TraP12114270[1]-027.AJ001.023 IRDIG544.12 93.3 95 170 w/TraP12114270[1]-027.AJ001.028 IRDIG544.12 96.7 85 160 w/TraP12114270[1]-027.AJ001.027 IRDIG544.12 98.3 86.7 150 w/TraP12114257[1]-016.AJ001.030 IRDIG544.12 100 86.7 100 114257[1]-016.AJ001.024IRDIG544.12 68.3 97 140 114257[1]-016.AJ001.021 IRDIG544.12 100 99.3 130114257[1]-016.AJ001.027 IRDIG544.12 100 71.7 130 114257[1]-016.AJ001.022IRDIG544.12 100 81.7 120 114267[1]-021.AJ001.039 IRDIG544.12 91.7 61.7230 w/TraP12 114267[1]-021.AJ001.034 IRDIG544.12 95 80 180 w/TraP12114267[1]-021.AJ001.044 IRDIG544.12 83.3 75 210 w/TraP12114268[1]-023.AJ001.036 IRDIG544.12 81 99 320 w/TraP12114268[1]-023.AJ001.041 IRDIG544.12 100 83.3 410 w/TraP12114268[1]-023.AJ001.034 IRDIG544.12 93.3 55 520 w/TraP12114268[1]-023.AJ001.039 IRDIG544.12 91.7 80 620 w/TraP12114268[1]-023.AJ001.026 IRDIG544.12 98.3 96 440 w/TraP12114268[1]-026.AJ001.053 IRDIG544.12 90 73.3 390 w/TraP12114268[1]-026.AJ001.046 IRDIG544.12 90 84.3 500 w/TraP12114268[1]-026.AJ001.037 IRDIG544.12 99.3 71.7 320 w/TraP12114268[1]-026.AJ001.038 IRDIG544.12 91.7 65 320 w/TraP12114268[1]-026.AJ001.052 IRDIG544.12 97.7 66.7 360 w/TraP12114271[1]-011.001AJ.031 IRDIG544.12 100 95 4 w/TraP12114271[1]-011.001AJ.042 IRDIG544.12 100 95 4 w/TraP12114271[1]-011.001AJ.043 IRDIG544.12 96 91.7 4 w/TraP12114271[1]-011.001AJ.047 IRDIG544.12 100 97.7 3 w/TraP12114271[1]-011.001AJ.046 IRDIG544.12 100 98 3 w/TraP12114270[1]-023.001AJ.050 IRDIG544.12 90 80 210 w/TraP12114270[1]-023.001AJ.055 IRDIG544.12 99.3 73.3 260 w/TraP12114270[1]-023.001AJ.044 IRDIG544.12 100 92 250 w/TraP12114270[1]-023.001AJ.054 IRDIG544.12 100 88.3 210 w/TraP12114270[1]-023.001AJ.058 IRDIG544.12 100 82.7 140 w/TraP12 B104 control100 100 0 B104 control 100 100 0 B104 control 100 100 0 B104 control 10099.3 0 B104 control 100 86 0

Reduced feeding damage caused by southwestern corn borer (SWCB) andsouthern armyworm (SAW) was observed when the insect larvae were exposedto plants containing truncated Cry1Ca modified protein, at a range ofprotein expression from 140-340 ng/cm² (Table 7). The average expressionwas 210 ng/cm² with a standard deviation of 35.

TABLE 7 Bioassay of IRDIG544.12 T₁ maize plants when fed to southwesterncorn borer (SWCB) and southern army worm (SAW) SWCB SAW Plant Name Avg.Dmg. Avg. Dmg. 112726[1]-015.AJ001.047 2 3.0 112726[1]-015.AJ001.030 43.0 112726[1]-015.AJ001.019 1 1.3 112726[1]-015.AJ001.034 2 2.0 YFPnegative control 98.3 94.3 YFP negative control 93.3 62.5 YFP negativecontrol 100 27.5 YFP negative control 92.7 67.5 YFP negative control97.7 30.0 YFP negative control 100 45.0 B104 98.3 94.3 B104 100 91.7B104 100 97.0 B104 65 70.0 B104 100 86.7

Field trials on corn borers were conducted at two locations: one inIndiana (IN), United States and the other in Mississippi (MS), UnitedStates. Multiple constructs and events were tested for each treatment.Cry1Ab and Cry1F served as positive controls in the ECB trials. The nullserved as a negative control.

To assess ECB efficacy, each plant received ten second-instar ECB larvaein the whorl of V6-V7 stage plants. In MS, Southwestern corn borer(SWCB) second-instar larvae were also artificially infested in thewhorls of V9 corn (22 larvae per plant). The ECB and SWCB used wereobtained from Benzon laboratory. In both ECB trials, plants wereevaluated 2 weeks after infestation for foliar damage (Guthrie 1-9scale) (Guthrie et al. 1960) where 1 is no visible injury and 9 is mostleaves with long lesions (Table 8). In the MS SWCB trials, the plantswere examined 4-5 days after the whorl rating for stalk damage and liveinsects. Data collected included number of tunnels per stalk, length oftunnels and live larvae/pupae per stalk.

TABLE 8 Corn Borer damage score criterion (whorl damage). ScoreCriterion 1 No visible leaf injury or small amount of shot hole typeinjury on a few leaves. 2 Small amount of shot-hole type lesions on afew leaves. 3 Shot-hole injury common on several leaves. 4 Severalleaves with shot-holes and elongated lesions. 5 Several leaves withelongated lesions. 6 Several leaves with elongated lesions (ca. 1 inch).7 Long lesions common on one-half of the leaves. 8 Long lesions commonon about ⅔ of the leaves. 9 Most leaves with long lesions.

ECB Field Trials. ECB whorl damage was measured for Cry1Ca activity andshowed significantly better whorl protection when compared to the null.Cry1Ca event activities were not statistically equivalent to the whorlprotection provided by Cry1Ab and Cry1F.

Data generated in MS further reinforced the unexpected high level ofplant protection for Cry1Ca. High feeding pressure was established inthis study. Significant control was measured for Cry1Ca when compared tofoliar and stalk damage on the null. Very few live insects were foundsurviving in the Cry1Ca stalks. Significant whorl and stalk protectionwas measured for Cry1Ab and Cry1F events when compared to the null.

TABLE 9 ECB Foliar Whorl Data, IN (Average Across Multiple Events) Avg.Whorl Damage Rating Range of Gene Events (1-9 rating) means Cry1Ca 42.31 B 2.00-2.65 Cry1Ab 8 1.00 A 1.00 Cry1F 12 1.03 A 1.00-1.20 Null 14.65 C Means followed by different letters are significantly different(P ≤ 0.05).

TABLE 10 ECB Foliar Whorl and Stalk Data, MS (Average Across MultipleEvents) Avg. Whorl Avg. # of Number Damage Avg. # Avg. Length Larvae +Toxin of Rating Tunnels (cm) of Pupae Gene Events (1-9) Per StalkTunnels Per Stalk Cry1Ca 3 2.74 B 0.53 B 1.43 B 0.25 B Cry1Ab 8 1.66 D0.00 C 0.00 C 0.00 C Cry1F 8 1.85 C 0.00 C 0.00 C 0.00 C Null 1 6.77 A1.93 A 9.15 A 1.89 A For all data columns, all gene events weresignificantly different from the null values (P < 0.05). Within eachcolumn, means followed by different letters are significantly different(P ≤ 0.05).

In the SWCB trial, only 2 events per B.t. were evaluated. High feedingpressure was established in this study. Statistically equivalent stalkprotection and number of larvae and pupae per stalk were measured forCry1Ab, Cry1F, and Cry1Ca events.

TABLE 11 SWCB Foliar Whorl and Stalk Data, MS Avg. Whorl Avg. # ofNumber Damage Avg. # Avg. Length Larvae + Toxin of Rating Tunnels (cm)of Pupae Gene Events (1-9) Per Stalk Tunnels Per Stalk Cry1Ca 2 1.90 C0.07 B 0.20 B 0.02 B Cry1Ab 2 1.91 C 0.00 B 0.00 B 0.00 B Cry1F 2 2.18 B0.07 B 0.54 B 0.03 B Null 1 7.17 A 3.53 A 28.62 A  3.06 A Within eachcolumn, means followed by different letters are significantly different(P ≤ 0.05).

The active form of Cry1Ca is composed of amino acids 29-628. The fulllength (1-1164), or cleaved forms (1-628 and 29-628) are active whenpresented to insects, since they are processed to the 29-628 form.

Example 5 Corn Earworm Field Trials

Field trials on corn earworm were conducted in Fowler, Ind. withmultiple constructs and events (SEQ ID NO:31). The null served as anegative control. Each plant received five first instar larvae in thegreen silks of corn ears. CEW were obtained from Benzon Laboratory. Tencorn ears per plot per event were evaluated to assess the level ofkernel damage in corn ears infested with CEW. All transgenic eventsprovided significantly lower levels of kernel damage when compared tothe null. There was significant suppression of larval feeding on Cry1Caplants (Table 12).

TABLE 12 CEW Kernel Consumption Data, IN Avg. area (cm²) % reduction inarea of kernel consumed kernels Gene Entries consumed* (compared tonull) Cry1Ca 4 1.72 B 63.2 Cry1F 12 1.86 B 60.3 Null 1 4.68 A

Example 6 Agrobacterium Transformation

Standard cloning methods were used in the construction of binary planttransformation and expression plasmids. Agrobacterium binary plasmidswhich contained the cry1Ca expression cassettes were engineered usingGateway® Technology (Invitrogen, Carlsbad, Calif.) and used inAgrobacterium-mediated plant transformation. Restriction endonucleaseswere obtained from New England BioLabs (NEB; Ipswich, Mass.) and T4 DNALigase (Invitrogen) were used for DNA ligation. Gateway reactions wereperformed using Gateway® LR Clonase® enzyme mix (Invitrogen) Plasmidpreparations were performed using the NucleoSpin® Plasmid Preparationkit or the NucleoBond® AX Xtra Midi kit (both from Macherey-Nagel),following the instructions of the manufacturers. DNA fragments werepurified using the QIAquick PCR Purification Kit or the QIAEX II GelExtraction Kit (both from Qiagen) after gel isolation.

DNA fragments comprising the nucleotide sequences that encode theinsecticidal proteins, or fragments thereof, were synthesized by acommercial vendor (e.g. DNA2.0, Menlo Park, Calif.) and supplied ascloned fragments in standard plasmid vectors, or were obtained bystandard molecular biology manipulation of other constructs containingappropriate nucleotide sequences. Unique restriction sites internal toeach gene was identified and a fragment of each gene synthesized, eachcontaining a specific deletion or insertion. The modified Cry fragmentswere subcloned into other Cry fragments at an appropriate restrictionsite to obtain a region encoding the desired full-length protein, fusedproteins, or deleted variant proteins.

Electro-competent cells of Agrobacterium tumefaciens strain Z707S (astreptomycin-resistant derivative of Z707; Hepburn et al., 1985) wereprepared and transformed using electroporation (Weigel and Glazebrook,2002). After electroporation, 1 mL of YEP broth (gm/L: yeast extract,10; peptone, 10; NaCl, 5) was added to the cuvette and the cell-YEPsuspension was transferred to a 15 mL culture tube for incubation at 28°C. in a water bath with constant agitation for 4 hours. The cells wereplated on YEP plus agar (25 gm/L) with spectinomycin (200 μg/mL) andstreptomycin (250 μg/mL) and the plates were incubated for 2-4 days at28° C. Well separated single colonies were selected and streaked ontofresh YEP+agar plates with spectinomycin and streptomycin as before, andincubated at 28° C. for 1-3 days.

The presence of the insecticidal protein gene insert in the binary planttransformation vector was performed by PCR analysis usingvector-specific primers with template plasmid DNA prepared from selectedAgrobacterium colonies. The cell pellet from a 4 mL aliquot of a 15 mLovernight culture grown in YEP with spectinomycin and streptomycin asbefore was extracted using Qiagen Spin Mini Preps, performed permanufacturer's instructions. Plasmid DNA from the binary vector used inthe Agrobacterium electroporation transformation was included as acontrol. The PCR reaction was completed using Taq DNA polymerase fromInvitrogen per manufacturer's instructions at 0.5× concentrations. PCRreactions were carried out in a MJ Research Peltier Thermal Cyclerprogrammed with the following conditions: Step 1) 94° C. for 3 minutes;Step 2) 94° C. for 45 seconds; Step 3) 55° C. for 30 seconds; Step 4)72° C. for 1 minute per kb of expected product length; Step 5) 29 timesto Step 2; Step 6) 72° C. for 10 minutes. The reaction was maintained at4° C. after cycling. The amplification products were analyzed by agarosegel electrophoresis (e.g. 0.7% to 1% agarose, w/v) and visualized byethidium bromide staining. A colony was selected whose PCR product wasidentical to the plasmid control.

TABLE 13 Description of plasmids for expressing DIG-465 and DIG-473 inmaize. Plasmid Description pDAB115752ZmUbi1/DIG-465/ZmPer5::SCBV(MAM)v2/ AAD-1v3/ZmLip pDAB115753ZmUbi1/DIG-473/ZmPer5::SCBV(MAM)v2/ AAD-1v3/ZmLip pDAB112725ZmUbi1/Cry1Ca(Zm)/ZmPer5::SCBV(MAM)/ AAD-1v3./ZmLip pDAB112726ZmUbi1/Cry1Ca(HGC)/ZmPer5::SCBV(MAM)/ AAD-1v3/ZmLip

Example 7 Production of DIG-465 and DIG-473 B.t. Insecticidal Proteinsand Variants in Monocot Plants

Agrobacterium-mediated transformation of maize. Seeds from a High II F₁cross (Armstrong et al., 1991) were planted into 5-gallon-potscontaining a mixture of 95% Metro-Mix 360 soilless growing medium (SunGro Horticulture, Bellevue, Wash.) and 5% clay/loam soil. The plantswere grown in a greenhouse using a combination of high pressure sodiumand metal halide lamps with a 16:8 hour Light:Dark photoperiod. Forobtaining immature F₂ embryos for transformation, controlledsib-pollinations were performed. Immature embryos were isolated at 8-10days post-pollination when embryos were approximately 1.0 to 2.0 mm insize.

Infection and co-cultivation. Maize ears were surface sterilized byscrubbing with liquid soap, immersing in 70% ethanol for 2 minutes, andthen immersing in 20% commercial bleach (0.1% sodium hypochlorite) for30 minutes before being rinsed with sterile water. A suspensionAgrobacterium cells containing a superbinary vector was prepared bytransferring 1-2 loops of bacteria grown on YEP solid medium containing100 mg/L spectinomycin, 10 mg/L tetracycline, and 250 mg/L streptomycinat 28° C. for 2-3 days into 5 mL of liquid infection medium (LS BasalMedium (Linsmaier and Skoog, 1965), N6 vitamins (Chu et al., 1975), 1.5mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), 68.5 gm/L sucrose, 36.0gm/L glucose, 6 mM L-proline, pH 5.2) containing 100 μM acetosyringone.The solution was vortexed until a uniform suspension was achieved, andthe concentration was adjusted to a final density of 200 Klett units,using a Klett-Summerson colorimeter with a purple filter. Immatureembryos were isolated directly into a micro centrifuge tube containing 2mL of the infection medium. The medium was removed and replaced with 1mL of the Agrobacterium solution with a density of 200 Klett units, andthe Agrobacterium and embryo solution was incubated for 5 minutes atroom temperature and then transferred to co-cultivation medium (LS BasalMedium, N6 vitamins, 1.5 mg/L 2,4-D, 30.0 gm/L sucrose, 6 mM L-proline,0.85 mg/L AgNO3, 100 μM Acetosyringone, 3.0 gm/L Gellan gum(PhytoTechnology Laboratories, Lenexa, Kans.), pH 5.8) for 5 days at 25°C. under dark conditions.

After co-cultivation, the embryos were transferred to selective mediumafter which transformed isolates were obtained over the course ofapproximately 8 weeks. For selection of maize tissues transformed with asuperbinary plasmid containing a plant expressible pat or bar selectablemarker gene, an LS based medium (LS Basal medium, N6 vitamins, 1.5 mg/L2,4-D, 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic acid monohydrate;PhytoTechnologies Labr.), 30.0 gm/L sucrose, 6 mM L-proline, 1.0 mg/LAgNO3, 250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) was used withBialaphos (Gold BioTechnology). The embryos were transferred toselection media containing 3 mg/L Bialaphos until embryogenic isolateswere obtained. Recovered isolates were bulked up by transferring tofresh selection medium at 2-week intervals for regeneration and furtheranalysis.

Regeneration and seed production. For regeneration, the cultures weretransferred to “28” induction medium (MS salts and vitamins, 30 gm/Lsucrose, 5 mg/L Benzylaminopurine, 0.25 mg/L 2, 4-D, 3 mg/L Bialaphos,250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) for 1 week underlow-light conditions (14 μEm-2 s-1) then 1 week under high-lightconditions (approximately 89 μEm-2 s-1). Tissues were subsequentlytransferred to “36” regeneration medium (same as induction medium exceptlacking plant growth regulators). When plantlets grew to 3-5 cm inlength, they were transferred to glass culture tubes containing SHGAmedium (Schenk and Hildebrandt salts and vitamins (1972);PhytoTechnologies Labr.), 1.0 gm/L myo-inositol, 10 gm/L sucrose and 2.0gm/L Gellan gum, pH 5.8) to allow for further growth and development ofthe shoot and roots. Plants were transplanted to the same soil mixtureas described earlier herein and grown to flowering in the greenhouse.Controlled pollinations for seed production were conducted.

The level of expression of DIG-465 by construct 115752 and the level ofexpression of DIG-473 by construct 115753 is presented in FIG. 1 . Bothexpressed similar levels of their respective proteins, at approximately70-80 ng/cm² measured in leaves using a leaf punch to obtain the tissuesample.

SDS-PAGE of extract was taken from maize expressing the gene thatencodes full length Cry1Ca protein (MR-1206) (mw 130 kDa). At least fiveprotein products were detected by immune-blotting using a polyclonalantibody directed against Cry1Ca. The full length (130 kDa) protein, asencoded by the gene inserted into maize was detected. The other bandsrepresent proteolytic products of this protein. A protein fragmentcomposed of amino acid sequences 1-628, representing the core toxin wasdetermined to have a molecular weight of 70 kDa. A 68 kDa bandrepresented a protein composed of amino acids 29-628, where the first 28amino acids from the N-terminus were deleted. The first three bands werefunctionally active against S. frugiperda and other lepidopteraninsects. A fourth band represented a cleaved protein composed of aminoacids 74-628 (mw 62 kDa), and a fifth band represented the Cry1Caprotein that was further processed to amino acids 74-596 (mw 59 kDa).The 62 kDa and 59 kDa bands were not functionally active against S.frugiperda and other lepidopteran insects, yet represent major proteinproducts.

Example 8 Bioassay of Transgenic Maize

Bioactivity of the DIG-465 and DIG-473 protein and variants produced inplant cells was demonstrated by methods known to those skilled in theart (see, for example Huang et al., 2006). Efficacy may be demonstratedby feeding various plant tissues or tissue pieces derived from a plantproducing the DIG-465 or DIG-473 protein or variants to target insectsin a controlled feeding environment. Alternatively, protein extracts maybe prepared from various plant tissues derived from a plant producingthe DIG-465 or DIG-473 protein or variants and incorporated in anartificial diet bioassay as previously described herein. It is to beunderstood that the results of such feeding assays are to be compared tosimilarly conducted bioassays that employ appropriate control tissuesfrom host plants that do not produce the DIG-465 or DIG-473 protein orvariants, or to other control samples.

The biological activity of various events produced in maize fromconstruct 115752 (DIG-465) were tested for preventing leaf damage causedby the feeding activity of either FAW or Cry1Fa resistant FAW (rFAW).The results show that events that expressed DIG-465 protein exhibitedless feeding damage than plants not expressing the protein, and that theeffect was dose dependent, with higher expression of DIG-465 resultingin less feeding damage caused by either FAW or rFAW, with the affectapparently greater against rFAW (Table 14 and FIG. 2 ).

Similarly, the biological activity of various events produced in maizefrom construct 115753 (DIG-473) were tested for preventing leaf damagecaused by the feeding activity of either FAW or Cry1Fa resistant FAW(rFAW). The results show that events that expressed DIG-473 proteinexhibited less feeding damage than plants not expressing the protein,and that the effect was dose dependent, with higher expression ofDIG-473 resulting in less feeding damage caused by either FAW or rFAW,with the affect apparently greater against rFAW (Table 14 and FIG. 3 ).

TABLE 14 FAW bioassay data when exposed to DIG-465, DIG-473, or controlsFAW rFAW Accumulated Avg. Avg. toxin Plant Name DIG # Dmg. Dmg. (ng/cm²)115752[1]-001.001 DIG-465 65 20 24 115752[1]-002.001 DIG-465 20 4.375 46115752[1]-003.001 DIG-465 32.5 3.75 75 115752[1]-004.001 DIG-465 10098.75 0.7 115752[1]-005.001 DIG-465 30 8.75 110 115752[1]-006.001DIG-465 40 11.25 44 115752[1]-007.001 DIG-465 22.5 5 110115752[1]-009.001 DIG-465 3.125 3.125 56 115752[1]-010.001 DIG-465 17.511.25 53 115752[1]-011.001 DIG-465 3.125 1.75 170 115752[1]-012.001DIG-465 27.5 5 16 115752[1]-013.001 DIG-465 20 1 61 115752[1]-014.001DIG-465 25 3.75 97 115752[1]-016.001 DIG-465 15 3.75 110115752[1]-017.001 DIG-465 75 45 2 115752[1]-018.001 DIG-465 25 4.375 72115752[1]-019.001 DIG-465 10 2.125 37 115752[1]-020.001 DIG-465 25 1 120115752[1]-021.001 DIG-465 20 1.75 65 115752[1]-022.001 DIG-465 12.5 7.5110 115752[1]-023.001 DIG-465 35 11.25 84 115752[1]-024.001 DIG-465 47.510 67 115752[1]-025.001 DIG-465 20 5 120 115752[1]-026.001 DIG-465 4.3758 130.00 115752[1]-027.001 DIG-465 5 3.125 56 115752[1]-028.001 DIG-46510 3.75 96 115752[1]-029.001 DIG-465 4.375 2.5 80 115752[1]-030.001DIG-465 8.75 10.625 21 115752[1]-031.001 DIG-465 100 100 0115753[1]-001.001 DIG-473 100 100 0 115753[1]-002.001 DIG-473 12.5 8.75130 115753[1]-003.001 DIG-473 20 7.5 78 115753[1]-004.001 DIG-473 203.125 110 115753[1]-005.001 DIG-473 25 11.875 24 115753[1]-006.001DIG-473 12.5 1.75 96 115753[1]-007.001 DIG-473 8.75 3.75 89115753[1]-008.001 DIG-473 25 3.375 130 115753[1]-010.001 DIG-473 20 6.2589 115753[1]-011.001 DIG-473 15 4.375 100 115753[1]-012.001 DIG-47313.75 2.125 79 115753[1]-013.001 DIG-473 17.5 2.75 54 115753[1]-014.001DIG-473 10 12.5 65 115753[1]-015.001 DIG-473 20 3.75 130115753[1]-016.001 DIG-473 7.5 3.125 49 115753[1]-017.001 DIG-473 2.1253.125 110 115753[1]-018.001 DIG-473 100 100 0.7 115753[1]-019.001DIG-473 100 100 0 115753[1]-020.001 DIG-473 11.875 1.75 90115753[1]-021.001 DIG-473 10 3.75 130 115753[1]-022.001 DIG-473 40 5 120115753[1]-023.001 DIG-473 7.5 3.75 110 115753[1]-025.001 DIG-473 13.753.375 72 115753[1]-026.001 DIG-473 12.5 4.375 150 115753[1]-027.001DIG-473 5.625 2.5 93 115753[1]-028.001 DIG-473 5 11.875 48115753[1]-029.001 DIG-473 3.125 3.125 56 115753[1]-030.001 DIG-473 7.53.125 120.00 B104 100 100 N/A B104 100 100 N/A B104 100 100 N/A YFPnegative control 100 100 N/A YFP negative control 100 100 N/A YFPnegative control 100 100 N/A B104 100 100 N/A B104 100 100 N/A B104 100100 N/A YFP negative control 100 100 N/A YFP negative control 100 100N/A YFP negative control 100 100 N/A

Example 9 Production of Bt Insecticidal Proteins and Variants in DicotPlants

Arabidopsis Transformation. Arabidopsis thaliana Col-01 was transformedusing the floral dip method (Weigel and Glazebrook, 2002). The selectedAgrobacterium colony was used to inoculate 1 mL to 15 mL cultures of YEPbroth containing appropriate antibiotics for selection. The culture wasincubated overnight at 28° C. with constant agitation at 220 rpm. Eachculture was used to inoculate two 500 mL cultures of YEP brothcontaining appropriate antibiotics for selection and the new cultureswere incubated overnight at 28° C. with constant agitation. The cellswere centrifuged at approximately 8700×g for 10 minutes at roomtemperature, and the resulting supernatant was discarded. The cellpellet was gently resuspended in 500 mL of infiltration mediacontaining: 1/2× Murashige and Skoog salts (Sigma-Aldrich)/Gamborg's B5vitamins (Gold BioTechnology, St. Louis, Mo.), 10% (w/v) sucrose, 0.044μM benzylaminopurine (10 μL/L of 1 mg/mL stock in DMSO) and 300 μL/LSilwet L-77. Plants approximately 1 month old were dipped into the mediafor 15 seconds, with care taken to assure submergence of the newestinflorescence. The plants were then laid on their sides and covered(transparent or opaque) for 24 hours, washed with water, and placedupright. The plants were grown at 22° C., with a 16:8 light:darkphotoperiod. Approximately 4 weeks after dipping, the seeds wereharvested.

Arabidopsis Growth and Selection Freshly harvested T₁ seed was allowedto dry for at least 7 days at room temperature in the presence ofdesiccant. Seed was suspended in a 0.1% agar/water (Sigma-Aldrich)solution and then stratified at 4° C. for 2 days. To prepare forplanting, Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.)in 10.5 inch×21 inch germination trays (T.O. Plastics Inc., Clearwater,Minn.) was covered with fine vermiculite, sub-irrigated with Hoagland'ssolution (Hoagland and Arnon, 1950) until wet, then allowed to drain for24 hours. Stratified seed was sown onto the vermiculite and covered withhumidity domes (KORD Products, Bramalea, Ontario, Canada) for 7 days.Seeds were germinated and plants were grown in a Conviron (ModelsCMP4030 or CMP3244; Controlled Environments Limited, Winnipeg, Manitoba,Canada) under long day conditions (16:8 light:dark photoperiod) at alight intensity of 120-150 μmol/m² sec under constant temperature (22°C.) and humidity (40-50%). Plants were initially watered with Hoagland'ssolution and subsequently with deionized water to keep the soil moistbut not wet.

The domes were removed 5-6 days post sowing and plants were sprayed witha chemical selection agent to kill plants germinated from nontransformedseeds. For example, if the plant expressible selectable marker geneprovided by the binary plant transformation vector was a pat or bar gene(Wehrmann et al., 1996), transformed plants may be selected by sprayingwith a 1000× solution of Finale (5.78% glufosinate ammonium, FarnamCompanies Inc., Phoenix, Ariz.). Two subsequent sprays were performed at5-7 day intervals. Survivors (plants actively growing) were identified7-10 days after the final spraying and were transplanted into potsprepared with Sunshine Mix LP5. Transplanted plants were covered with ahumidity dome for 3-4 days and placed in a Conviron incubator under theabove-mentioned growth conditions.

Those skilled in the art of dicot plant transformation will understandthat other methods of selection of transformed plants are available whenother plant expressible selectable marker genes (e.g. herbicidetolerance genes) are used.

Example 10 Transgenic Glycine max Comprising DIG Protein

Ten to 20 transgenic T₀ Glycine max plants harboring expression vectorsfor nucleic acids comprising Cry1Ca protein were generated as is knownin the art, including for example by Agrobacterium-mediatedtransformation. Mature soybean (Glycine max) seeds were sterilizedovernight with chlorine gas for sixteen hours. Following sterilizationwith chlorine gas, the seeds were placed in an open container in aLAMINAR™ flow hood to dispel the chlorine gas. Next, the sterilizedseeds were imbibed with sterile H₂O for sixteen hours in the dark usinga black box at 24° C.

Preparation of split-seed soybeans. The split soybean seed comprising aportion of an embryonic axis protocol required preparation of soybeanseed material which was cut longitudinally, using a #10 blade affixed toa scalpel, along the hilum of the seed to separate and remove the seedcoat, and to split the seed into two cotyledon sections. Carefulattention was made to partially remove the embryonic axis, wherein about½-⅓ of the embryo axis remained attached to the nodal end of thecotyledon.

Inoculation. The split soybean seeds comprising a partial portion of theembryonic axis were then immersed for about 30 minutes in a solution ofAgrobacterium tumefaciens (e.g., strain EHA 101 or EHA 105) containingbinary plasmid comprising DIG protein. The Agrobacterium tumefacienssolution was diluted to a final concentration of λ=0.6 OD₆₅₀ beforeimmersing the cotyledons comprising the embryo axis.

Co-cultivation. Following inoculation, the split soybean seed wasallowed to co-cultivate with the Agrobacterium tumefaciens strain for 5days on co-cultivation medium (Wang, Kan. Agrobacterium Protocols. 2. 1.New Jersey: Humana Press, 2006. Print.) in a Petri dish covered with apiece of filter paper.

Shoot induction. After 5 days of co-cultivation, the split soybean seedswere washed in liquid Shoot Induction (SI) media consisting of B5 salts,B5 vitamins, 28 mg/L Ferrous, 38 mg/L Na₂EDTA, 30 g/L sucrose, 0.6 g/LMES, 1.11 mg/L BAP, 100 mg/L TIMENTIN™, 200 mg/L cefotaxime, and 50 mg/Lvancomycin (pH 5.7). The split soybean seeds were then cultured on ShootInduction I (SI I) medium consisting of B5 salts, B5 vitamins, 7 g/LNoble agar, 28 mg/L Ferrous, 38 mg/L Na₂EDTA, 30 g/L sucrose, 0.6 g/LMES, 1.11 mg/L BAP, 50 mg/L TIMENTIN™, 200 mg/L cefotaxime, 50 mg/Lvancomycin (pH 5.7), with the flat side of the cotyledon facing up andthe nodal end of the cotyledon imbedded into the medium. After 2 weeksof culture, the explants from the transformed split soybean seed weretransferred to the Shoot Induction II (SI II) medium containing SI Imedium supplemented with 6 mg/L glufosinate (LIBERTY®).

Shoot elongation. After 2 weeks of culture on SI 11 medium, thecotyledons were removed from the explants and a flush shoot padcontaining the embryonic axis were excised by making a cut at the baseof the cotyledon. The isolated shoot pad from the cotyledon wastransferred to Shoot Elongation (SE) medium. The SE medium consisted ofMS salts, 28 mg/L Ferrous, 38 mg/L Na₂EDTA, 30 g/L sucrose and 0.6 g/LMES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid, 0.1 mg/L IAA, 0.5mg/L GA3, 1 mg/L zeatin riboside, 50 mg/L TIMENTIN™, 200 mg/Lcefotaxime, 50 mg/L vancomycin, 6 mg/L glufosinate, 7 g/L Noble agar,(pH 5.7). The cultures were transferred to fresh SE medium every 2weeks. The cultures were grown in a CONVIRON™ growth chamber at 24° C.with an 18 h photoperiod at a light intensity of 80-90 μmol/m² sec.

Rooting. Elongated shoots which developed from the cotyledon shoot padwere isolated by cutting the elongated shoot at the base of thecotyledon shoot pad, and dipping the elongated shoot in 1 mg/L IBA(Indole 3-butyric acid) for 1-3 minutes to promote rooting. Next, theelongated shoots were transferred to rooting medium (MS salts, B5vitamins, 28 mg/L Ferrous, 38 mg/L Na₂EDTA, 20 g/L sucrose and 0.59 g/LMES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid 7 g/L Noble agar,pH 5.6) in phyta trays.

Cultivation. Following culture in a CONVIRON™ growth chamber at 24° C.,18 h photoperiod, for 1-2 weeks, the shoots which had developed rootswere transferred to a soil mix in a covered sundae cup and placed in aCONVIRON™ growth chamber (models CMP4030 and CMP3244, ControlledEnvironments Limited, Winnipeg, Manitoba, Canada) under long dayconditions (16 hours light/8 hours dark) at a light intensity of 120-150μmol/m² sec under constant temperature (22° C.) and humidity (40-50%)for acclimatization of plantlets. The rooted plantlets were acclimatedin sundae cups for several weeks before they were transferred to thegreenhouse for further acclimatization and establishment of robusttransgenic soybean plants.

Development and morphological characteristics of transgenic lines werecompared with nontransformed plants. Plant root, shoot, foliage andreproduction characteristics were compared. There were no observabledifference in root length and growth patterns of transgenic andnontransformed plants. Plant shoot characteristics such as height, leafnumbers and sizes, time of flowering, floral size and appearance weresimilar. In general, there were no observable morphological differencesbetween transgenic lines and those without expression of DIG proteinswhen cultured in vitro and in soil in the glasshouse.

Example 11 Transformation of Additional Crop Species

Cotton is transformed with B.t. proteins (with or without a chloroplasttransit peptide) to provide control of lepidopteran insects by utilizinga method known to those of skill in the art, for example, substantiallythe same techniques previously described in EXAMPLE 9 of U.S. Pat. No.7,838,733, or Example 12 of PCT International Patent Publication No. WO2007/053482.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. With the teachingsprovided herein, one skilled in the art could readily produce and usethe various toxins and polynucleotide sequences described herein.

1. A liquid formulation or bait granule comprising a modified Cry1Catoxin comprising residues 2 to 68 of SEQ ID NO:2 wherein amino acidresidue 54 of SEQ ID NO: 2 is Ala, amino acid residue 57 of SEQ ID NO: 2is selected from the group consisting of Leu and Met, and amino acidresidue 68 of SEQ ID NO: 2 is selected from the group consisting of Val,Phe, and Ile.
 2. A liquid formulation or bait granule comprising amodified Cry1Ca toxin comprising residues 2 to 628 of SEQ ID NO:10wherein amino acid residue 54 is Ala, amino acid residue 57 is selectedfrom the group consisting of Leu and Met, amino acid residue 68 isselected from the group consisting of Val, Phe, and Ile, amino acidresidue 73 is selected from the group consisting of Trp, Ala and Met,amino acid residue 596 is selected from the group consisting of Phe, Metand Ala, and amino acid residue 620 is selected from the groupconsisting of Leu and Phe.
 3. The liquid formulation or bait granule ofclaim 1 further comprising to said modified Cry1Ca toxins a carboxyterminal extension consisting of amino acid residues 629 to 1164 of SEQID NO:36.
 4. The liquid formulation or bait granule of claim 2 furthercomprising to said modified Cry1Ca toxins a carboxy terminal extensionconsisting of amino acid residues 629 to 1164 of SEQ ID NO:36.
 5. Theliquid formulation or bait granule of claim 1 further comprising to saidmodified Cry1Ca toxins an amino terminal extension consisting of aminoacid residues 1 to 74 of SEQ ID NO:40.
 6. The liquid formulation or baitgranule of claim 2 further comprising to said modified Cry1Ca toxins anamino terminal extension consisting of amino acid residues 1 to 74 ofSEQ ID NO:40.
 7. The liquid formulation or bait granule of claim 3further comprising to said modified Cry1Ca toxins an amino terminalextension consisting of amino acid residues 1 to 74 of SEQ ID NO:40. 8.The liquid formulation or bait granule of claim 4 further comprising tosaid modified Cry1Ca toxins an amino terminal extension consisting ofamino acid residues 1 to 74 of SEQ ID NO:40.
 9. A liquid formulation orbait granule comprising a modified Cry1Ca toxin chosen from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQID NO:40.
 10. (canceled)
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 13. (canceled)14. (canceled)
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 17. (canceled) 18.(canceled)
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 25. A method of controlling plant insectpests which comprises allowing susceptible pests to feed on said liquidformulation or bait granule of claim
 1. 26. A method of controllingplant insect pests that have developed resistance to other Cry toxinswhich comprises allowing susceptible pests to feed on said liquidformulation or bait granule of claim 2.